Heterodimeric proteins and methods for producing and purifying them

ABSTRACT

The present invention relates to engineered heteromultimeric proteins, and more specifically, to methods for producing and purifying heterodimeric proteins, such as bispecific antibodies and other heterodimeric proteins comprising immunoglobulin-like hinge sequences. Methods for producing and purifying such engineered heterodimeric proteins and their use in diagnostics and therapeutics are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/351,275, filed Nov. 14, 2016, which is a divisional of U.S.application Ser. No. 13/697,683, filed on Jan. 17, 2013, now U.S. Pat.No. 9,527,926, which is a National Stage Application under 35 U.S.C. §371 of PCT/US2011/036419, filed on May 13, 2011, which claims thebenefits of U.S. Provisional Application No. 61/345,047 filed May 14,2010, and U.S. Provisional Application No. 61/485,097 filed May 11,2011, which are both hereby incorporated by reference in theirentireties.

REFERENCE TO SEQUENCE LISTING

This application is being filed electronically via EFS-Web and includesan electronically submitted sequence listing in .txt format. The .txtfile contains a sequence listing entitled“PC71659C_SequenceListing_ST25.txt” created on Oct. 10, 2018, and havinga size of 18 KB. The sequence listing contained in this .txt file ispart of the specification and is incorporated herein by reference in itsentirety.

FIELD

The present invention relates to engineered heteromultimeric proteins,and more specifically, to methods for producing and purifyingheterodimeric proteins, such as bispecific antibodies and otherheterodimeric proteins comprising immunoglobulin-like hinge sequences.Methods for producing and purifying such engineered heterodimericproteins and their use in diagnostics and therapeutics are alsoprovided.

BACKGROUND

Antibodies possess a variety of properties which make them useful astherapeutic molecules. In addition to their ability to bind with highaffinity to a molecular target inside or outside of cells with highspecificity and selectivity, antibodies render their targeted bindingpartners susceptible to Fc-receptor cell-mediated phagocytosis andkilling through effector functions, such as complement induced pathwaysand ADCC (antibody-dependent cell-mediated cytotoxicity) relatedactivities.

Further, antibodies may be engineered in a variety of ways to furtherincrease their therapeutic utility. Antibodies having extended in vivohalf-lives, for example, may be produced by engineering Fc fusionmolecules, by treatment with biocompatible polymers such as polyethyleneglycol (PEG), or “pegylation” and by other engineering methods wellknown in the art. Antibodies have binding specificities for at least twodifferent antigens, called bispecific antibodies (BsAbs), have also beenengineered. See Nolan, O. and R. O'Kennedy (1990) Biochim Biophvs Acta1040(1): 1-1 1.; de Leij, L. et al., Adv Drug Deliv Rev 31(1-2): 105-129(1998); and Carter, P. J Immunol Methods 248(1-2): 7-15 (2001)). Whileclassical antibodies have identical sequences in each of the two arms(containing the antigen binding sites of Fab region) of the Y-shapedmolecule, bispecific antibodies have different sequences in each of thetwo Fab regions so that each arm of the Y-shaped molecule binds to adifferent antigen or epitope.

By being able to bind two different antigenic molecules or differentepitopes, BsAbs offer a wide variety of clinical applications astargeting agents for in vitro and in vivo diagnostics andimmunotherapies. In diagnostic areas, BsAbs have been used, e.g., tostudy functional properties of cell surface molecules, different Fcreceptors and their ability to mediate cytotoxicity (Fanger et al.,Crit. Rev. Immunol. 12:101-124 (1992); Nolan et al., Biochem. Biophys.Acta. 1040:1-11 (1990); and to immobilize enzymes and other agents toproduce immunodiagnostic and immunoassay reagents and methods.

Bispecific antibodies can also be used for in vitro or in vivo diagnosesof various disease states, including cancer (Songsivilai et al., Clin.Exp. Immunol. 79:315 (1990)). For example, one arm of the BsAb can beengineered to bind a tumor-associated antigen and the other arm to binda detectable marker. (See, e.g., Le Doussal et al., J. Nucl. Med.34:1662-1671 (1993), in which a BsAb having one arm which bound acarcinoembryonic antigen (CEA) and another arm which bound DPTA was usedfor radioimmunodetection of colorectal and thyroid carcinomas. See alsoStickney et al., Cancer Res. 51:6650-6655 (1991), describing a strategyfor detecting colorectal cancers expressing CEA by radioimmunodetection.

The use of bispecific antibodies for immunotherapy of cancer has beenreviewed (see e.g., Nolan and O'Kennedy 1990, supra; de Leij et al.(1998) supra; and Carter, P. (2001) supra.) BsAbs can be used to directa patient's cellular immune defense mechanisms specifically to a tumorcell or an infectious agent (e.g., virally infected cells such as HIV orinfluenza virus; protozoa such as Toxoplasma gondii). In particular, onecan redirect immune modulated cytotoxicity by engineering one arm of theBsAb to bind to a desired target (e.g. tumor cell or pathogen) and theother arm of the BsAb to bind to a cytotoxic trigger molecule, such asthe T-cell receptor or a Fc gamma receptor (thereby activatingdownstream immune effector pathways). Using this strategy, BsAbs whichbind to the Fc gamma RIII have been shown to mediate tumor cell killingby natural killer (NK) cell/large granular lymphocyte (LGL) cells invitro and to prevent tumor growth in vivo. (See, e.g., Segal et al.,Chem. Immunol. 47:179 (1989); Biologic Therapy of Cancer 2(4) DeVita etal. eds. J. B. Lippincott, Philadelphia (1992) p. 1.) In anotherexample, a bispecific antibody having one arm that binds Fc gamma RIIIand another that binds the HER2 receptor was developed for treatment oftumors that overexpress HER2 antigen (Hseih-Ma et al. Cancer Research52:6832-6839 (1992); and Weiner et al. Cancer Research 53:94-100(1993)). See also Shalaby et al., J. Exp. Med. 175(1):217 (1992) inwhich a fully humanized F(ab′)2 BsAb comprising anti-CD3 linked toanti-p185(HER2) was used to target T cells to kill tumor cells thatoverexpress HER2 receptor.

Use of bispecific antibodies has been hindered by difficulties inobtaining BsAbs in sufficient quantity and purity. Traditionally, BsAbswere made using hybrid-hybridoma technology (Millstein and Cuello,Nature 305:537-539 (1983)). Methods for making BsAbs by chemicalcoupling have since been described (see, e.g., Shalaby et al., J. Exp.Med. 175:217-225 (1992); Rodrigues et al., Int. J. Cancers (Suppl.)7:45-50 (1992); Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).Diabody technology described by Hollinger et al., Proc. Natl. Acad. Sci.USA 90:6444-6448 (1993) has provided alternative procedures for makingBsAb fragments; as has the use of single chain Fv (sFv) dimers (see,e.g., Gruber et al., J. Immunol. 152: 5368 (1994).

To produce multispecific (e.g., bispecific) antibody heteromultimers(e.g., heterodimers), it is desirable to use methods that favorformation of the desired heteromultimer over homomultimer(s). One methodfor obtaining Fc-containing BsAbs remains the hybrid hybridomatechnique, in which two antibodies are co-expressed (Milstein andCuello, Nature 305:537-540 (1983); see Suresh, M. R., et al. MethodsEnzymol 121:210-228 (1986)). However, it is often inefficient withrespect to yield and purity, the desired heteromultimer often beingdifficult to further purify. Other techniques to favor heteromultimerformation have been described and involve engineering stericallycomplementary mutations in multimerization domains at the CH3 domaininterface, referred to as a “knobs-into-holes” strategy (see e.g.,Ridgway et al., Protein Eng. 9:617-621 (1996); Merchant et al., Nat.Biotechnol. 16(7): 677-81 (1998); see also U.S. Pat. Nos. 5,731,168 and7,183,076). Techniques involving replacing one or more residues thatmake up the CH3-CH3 interface in both CH3 domains with a charged aminoacid for promoting the heterodimer formation have also been described.WO2009/089004.

It would be desirable to find new methods for engineering bispecificantibody fragments and/or full length BsAbs, such as those which enablethe BsAbs to be expressed and recovered directly or efficiently fromrecombinant cell culture and/or which may be produced with efficientyields and purities, or having increased stability compared tobispecific antibodies in the art.

SUMMARY

In one aspect, this invention provides a heteromultimeric (e.g.,heterodimeric) protein comprising a hinge region, wherein the hingeregion comprises a first immunoglobulin-like hinge polypeptide and asecond immunoglobulin-like hinge polypeptide which interact together toform a dimeric hinge interface, wherein electrostatic interactionsbetween one or more charged amino acids within the hinge interface favorinteraction between the first and second hinge polypeptides overinteraction between two first hinge polypeptides or two second hingepolypeptides, thereby promoting heterodimer formation over homodimerformation. In some embodiments, the hinge region is an IgG hinge region.In some embodiments, the hinge region is an IgG1, IgG2, IgG3, or IgG4hinge region. In some embodiments, the IgG hinge region comprises ahuman IgG hinge region (e.g., human IgG1, IgG2, IgG3, or IgG4 hingeregion).

In some embodiments, the first hinge polypeptide comprises at least oneamino acid modification relative to a wild-type (WT) hinge region (e.g.,IgG hinge region), wherein the wild-type amino acid is replaced with anamino acid having an opposite charge to the corresponding amino acid inthe second hinge polypeptide.

In some embodiments, the first hinge polypeptide comprises at least oneamino acid modification relative to a wild-type hinge region (e.g., IgGhinge region), and the second hinge polypeptide comprises at least oneamino acid modification relative to a wild-type hinge region (e.g., IgGhinge region) in proximity to or at the same position as the amino acidmodification in the first hinge polypeptide, wherein the wild-type aminoacid in the second hinge polypeptide is replaced with an amino acidhaving an opposite charge to the corresponding amino acid in the firsthinge polypeptide. In some embodiments, the amino acid modifications canbe charged residues (e.g., Lys, Arg, His, Glu, and Asp) or polarresidues (e.g., Ser and Thr). In some embodiments, the first hingepolypeptide comprises a human IgG1 and the amino acid modification inthe first hinge polypeptide is at a position selected from the groupconsisting of 221 and 228. In some embodiments, the first hingepolypeptide comprises a human IgG2 and the amino acid modification inthe first hinge polypeptide is at a position selected from the groupconsisting of 223, 225, and 228. In some embodiments, the first hingepolypeptide comprises a human IgG4, and the amino acid modification inthe first hinge polypeptide is at position 228.

In other embodiments, the heterodimeric protein of the invention furthercomprises a CH3 region, wherein the CH3 region comprises a first CH3polypeptide and a second CH3 polypeptide which interact together to forma CH3 interface, wherein one or more amino acids within the CH3interface destabilize homodimer formation and are not electrostaticallyunfavorable to homodimer formation.

In some embodiments, the heteromultimeric (e.g. heterodimeric) proteinof the invention can be, for example, an antibody, a maxibody, amonobody, a peptibody, and an Fc fusion protein. In some embodiments,the heterodimeric protein is a bispecific antibody. In some embodiments,the heterodimeric protein is monospecific monovalent, bispecificmonovalent, or bispecific bivalent (e.g., monospecific monovalentantibody, bispecific monovalent, or bispecific bivalent antibody).

In another aspect, this invention provides a strategy for enhancing theformation of a desired heteromultimeric or heterodimeric protein (e.g.,bispecific antibody) by altering or engineering an interface between afirst and a second immunoglobulin-like Fc region (e.g., a hinge regionand/or a CH3 region). In some embodiments, one or more residues thatmake up the hinge interface are replaced with charged residues such thatthe electrostatic interactions between these charged residueselectrostatically favor heterodimer formation over homodimer formation.In further embodiments, one or more residues that make up the CH3interface are further replaced with charged residues such that theinteractions between the CH3 interface further promotes heterodimerformation over homodimer formation. In some embodiments, the engineeredCH3 interface destabilizes homodimer formation. In some embodiments, theengineered CH3 interface is not electrostatically unfavorable tohomodimer formation. In some embodiments, the engineered CH3 interfacesterically favors heterodimer formation over homodimer formation. Insome embodiments, the engineered CH3 interface electrostatically favorheterodimer formation over homodimer formation.

In another aspect, disclosed herein are heteromultimeric (e.g., aheterodimeric) proteins comprising an immunoglobulin-like CH3 regioncomprising a first CH3 polypeptide and a separate second CH3 polypeptidethat interact together to form a CH3 interface, wherein one or moreamino acids within the CH3 interface destabilize homodimer formation andare not electrostatically unfavorable to homodimer formation. In someembodiments, the first CH3 polypeptide comprises an amino acidmodification relative to a wild-type CH3 region sequence. In someembodiments, the first CH3 polypeptide further comprises a second aminoacid modification relative to a wild-type CH3 sequence. In someembodiments, the first CH3 polypeptide further comprises a third aminoacid modification relative to a wild-type CH3 sequence. In someembodiments, the second CH3 polypeptide comprises an amino acidmodification relative to a wild-type CH3 region sequence. In someembodiments, the second CH3 polypeptide further comprises a second aminoacid modification relative to a wild-type CH3 region sequence. In someembodiments, the second CH3 polypeptide further comprises a third aminoacid modification relative to a wild-type CH3 region sequence.

In some embodiments, the CH3 region is an IgG1, IgG2, IgG3, or IgG4 CH3region. In some embodiments, the CH3 region comprises a human IgG CH3region (e.g., human IgG1, IgG2, IgG3, or IgG4 CH3 region).

In some embodiments, the amino acid modification in the CH3 polypeptideis an amino acid substitution at a position selected from the groupconsisting of 349, 368, 405 and 409. In some embodiments, the amino acidmodification is selected from the group consisting of K409R, L368E, andL368D.

In some embodiments, an amino acid modification in the first CH3polypeptide is K409R and an amino acid modification in the second CH3polypeptide is L368E or L368D.

In another aspect, this invention also provides a method of producing aheteromultimeric, (e.g., heterodimeric) protein of the inventioncomprising the steps of: a) culturing a host cell comprising a nucleicacid molecule encoding the first polypeptide and a nucleic acid moleculeencoding the second polypeptide (the first and second polypeptidesexpressed from the same or from one or more different nucleic acidmolecules), wherein the cultured host cell expresses the first andsecond polypeptides; and b) optionally, recovering the heterodimericprotein from the host cell culture.

In another aspect, this invention also provides a method of producing aheteromultimeric, (e.g., heterodimeric) protein of the inventioncomprising the steps of: a) expressing the first polypeptide in a firsthost cell; b) expressing the second polypeptide in a second host cell;c) optionally, isolating the first polypeptide and the secondpolypeptide; and d) incubating the two polypeptides under a conditionsuitable for dimerization (for example, using a reducing agent such as,e.g., glutathione) to produce the heterodimeric protein.

In another aspect, this invention provides a method of purifying aheterodimeric protein comprising one or more Fc regions (e.g., a hingeregion and/or a CH3 region) which electrostatically favor heterodimerformation over homodimer formation.

In another aspect, this invention also provides methods of purifying aheterodimeric protein comprising an immunoglobulin-like Fc region andthe purification comprises at least one step that is based ondifferences in electrostatic interaction in the Fc regions. Theheterodimeric protein that can be purified by the methods of thisinvention may comprise an immunoglobulin-like hinge region and/orconstant region (e.g., CH2 region or CH3 region).

In some embodiments, the method comprises at least one step that isbased on differences in electrostatic interaction in the hinge region.In some embodiments, the method comprises at least one step that isbased on differences in electrostatic interaction in the constantregion. In some embodiments, the constant region can be a heavy chainconstant region, a CH2 region, or a CH3 region. In some embodiments, themethod comprises a chromatography step (e.g., ion exchangechromatography).

In another aspect, this invention provides polypeptides, nucleic acids,vectors and host cells that relate to the production of aheteromultimeric (e.g., heterodimeric) protein of the invention. Thisinvention also provides pharmaceutical compositions/formulations thatcomprise a heteromultimeric, e.g., heterodimeric protein of theinvention and methods of using such compositions.

In another aspect, a method of treating a condition, disorder or diseasein a subject is provided, the method comprising administering to thesubject an effective amount of a pharmaceutical composition comprising aheteromultimeric (e.g., heterodimeric) protein of the invention.

In another aspect, this invention also provides a polypeptide comprisingan immunoglobulin-like hinge polypeptide, wherein the hinge polypeptidecomprises at least one amino acid modification relative to a wild-typeimmunoglobulin-like hinge polypeptide, wherein the polypeptide hasincreased ability to form a heterodimeric protein with a secondpolypeptide, compared to a polypeptide comprising the wild-typeimmunoglobulin-like hinge polypeptide.

In another aspect, this invention also provides a polypeptide comprisingan CH3 polypeptide, wherein the CH3 polypeptide comprises at least oneamino acid modification relative to a wild-type CH3 polypeptide, whereinthe polypeptide has increased ability to form a heterodimeric proteinwith a second polypeptide, compared to a polypeptide comprising thewild-type CH3 hinge polypeptide. In some embodiments, the amino acidmodification is selected from the group consisting of K409R, L368E, andL368D.

In another aspect of the invention, the heterodimeric protein (e.g.,bispecific antibody) as described herein comprises a full-length humanantibody, wherein a first antibody variable domain of the heterodimericprotein is capable of recruiting the activity of a human immune effectorcell by specifically binding to an effector antigen located on the humanimmune effector cell, wherein a second antibody variable domain of theheterodimeric protein is capable of specifically binding to a targetantigen. In some embodiments, the human antibody has an IgG1, IgG2,IgG3, or IgG4 isotype.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an exemplary antibody mutant to describe the nomenclaturesystem used by this application to describe a heterodimeric protein.

FIG. 2 depicts human IgG2ΔA Fc region sequence (SEQ ID NO depicts 1).

FIG. 3 depicts a vector map for pCi.db.3×FLAG (or HA).Ab1.hFc1 vector.

FIG. 4 depicts human IgG1 wild-type Fc region sequence (SEQ ID NOdepicts 11).

FIG. 5 depicts human IgG4 wild-type Fc region sequence (SEQ ID NOdepicts 12).

FIG. 6A depicts an alignment of human IgG1 (EPKSCDKTHCPPCP (SEQ ID NO:57)), IgG2 (ERKCCVECPPCP (SEQ ID NO: 56)), and IgG4 (ESKYGPPCPSCP (SEQID NO: 55)) hinge regions.

FIG. 6B depicts a table of human IgG 4 and IgG1 mutants.

FIG. 6C depicts a table of human IgG2 mutants.

FIGS. 7A-7B depict an analysis of bispecific antibody formation fromIgG4 mutants.

FIGS. 8A-8B depict an analysis of bispecific antibody formation fromIgG2 mutants.

FIGS. 9A-9B depict results from a screen for IgG2 hinge mutations inK409R background.

FIG. 10 depicts an alignment of human IgG4 (SEQ ID NO: 60), IgG2ΔA (SEQID NO: 59), and IgG1 (SEQ ID NO: 58) CH3 regions.

FIGS. 11A-11B depicts results for “Glu” scanning on various human IgG4mutants.

FIGS. 12A-12B depict results for “Glu” scanning on various human IgG2mutants.

FIGS. 13A-13B depict mutations in both hinge regions and CH3 regions areimportant for heterodimer/bispecific antibody formation of human IgG2mutants.

FIGS. 14A-14B depicts results for “Glu” scanning on various human IgG1mutants.

FIGS. 15A-15B depict a comparison of bispecific antibody formationbetween different IgG isotypes.

FIGS. 16A-16C depict ion exchange elution profiles of hIgG1 mutants.Dashed line represents Ab1 antibody with 221R and 228R mutations in thehinge region and 409R mutation in the CH3 domain of the heavy chain.Dotted line represents Ab2 hIgG1 antibody with 221E and 228E mutationsin the hinge and 368E in the CH3 domain. Solid line represents elutionprofile of the Ab1-Ab2 bispecific antibody reaction products formedafter incubation of the Ab1 and Ab2 variants with 1 mM glutathione.

FIGS. 17A-17C depict ion exchange elution profiles of hIgG2 mutants.Dashed line represents Ab1 antibody with 223R, 225R, and 228R mutationsin the hinge region and 409R mutation in the CH3 domain of the heavychain. Dotted line represents Ab2 hIgG1 antibody with 223E, 225E, and228E mutations in the hinge and 368E in the CH3 domain. Solid linerepresents elution profile of the Ab1-Ab2 bispecific antibody reactionproducts formed after incubation of the Ab1 and Ab2 variants with 2 mMglutathione.

FIGS. 18A-18C depict ion exchange elution profiles of hIgG2 mutants. Thehinge mutations were exchanged relative to constructs shown in FIG.17A-17C resulting in two antibodies with less total charge difference.The pairing of the hinge mutations with different variable domains hasno effect on bispecific antibody formation. Dashed line represents Ab1antibody with 223E, 225E, and 228E mutations in the hinge region and409R mutation in the CH3 domain of the heavy chain. Dotted linerepresents Ab2 hIgG1 antibody with 223R, 225R, and 228R mutations in thehinge and 368E in the CH3 domain. Solid line represents elution profileof the Ab1-Ab2 bispecific antibody reaction products formed afterincubation of the Ab1 and Ab2 variants with 2 mM glutathione.

FIGS. 19A-19D depict co-expression of modified hinge polypeptides withlight chain sequences produces bispecific antibodies. Dash-dot-dot linerepresents ion exchange chromatography trace of bispecific antibodiesformed by co-expressing hIgG2.RRR.K409R.Ab1 heavy chain withhIgG2.EEE.L368E.Ab2 heavy chain, and Ab2 light chain. Solid linerepresents elution profile of the Ab1-Ab2 bispecific antibody reactionproducts formed after incubation of the purified Ab1 (223R, 225R, 228R,and 409R) heavy chain with Ab2 light chain and Ab2 (223E, 225E, 228E,and 368E) with 2 mM glutathione. Dotted line represents control antibodyAb2 hIgG2 with 223E, 225E, 228E, and 368E mutations. Dashed linerepresents control Ab1 antibody with 223R, 225R, 228R, and 409Rmutations in the heavy chain expressed together with Ab2 light chain.

FIGS. 20A-20D depict co-expression of modified hinge polypeptides withlight chain sequences produces bispecific antibodies. Dash-dot-dot linerepresents ion exchange chromatography trace of bispecific antibodiesformed by co-expressing hIgG1.RR.K409R.Ab1 heavy chain withhIgG1.EE.L368E.Ab2 heavy chain, and Ab2 light chain. Solid linerepresents elution profile of the Ab1-Ab2 bispecific antibody reactionproducts formed after incubation of the purified Ab1 (221R, 228R, and409R) heavy chain with Ab2 light chain and Ab2 (221E, 228E, and 368E)with 1 mM glutathione. Dotted line represents control antibody Ab2 hIgG1with 221E, 228E, and 368E mutations. Dashed line represents control Ab1antibody with 221R, 228R, and 409R mutations in the heavy chainco-expressed together with Ab2 light chain.

FIGS. 21A-21C depict the method of the invention does not depend on theidentity of the variable domains and is thus widely applicable. Dashedline represents Ab4 antibody with 221R and 228R mutations in the hingeregion and 409R mutation in the CH3 domain of the heavy chain. Dottedline represents Ab3 hIgG1 antibody with 221E and 228E mutations in thehinge and 368E in the CH3 domain. Solid line represents elution profileof the Ab4-Ab3 bispecific antibody reaction products formed afterincubation of the Ab4 and Ab3 variants with 1 mM glutathione.

FIGS. 22A-22C depict ion exchange chromatography can be used to separateIgG1 mutant (221R, 228R, and 409R) from IgG1 (221E, 228E, and 368E)mutant even if the variable domains are the same. The heterodimer ofthese two variants can also be separated from the homodimers using ionexchange chromatography. Dashed line represents Ab4 antibody with 221Rand 228R mutations in the hinge region and 409R mutation in the CH3domain of the heavy chain. Dotted line represents Ab4 hIgG1 antibodywith 221E and 228E mutations in the hinge and 368E in the CH3 domain.Solid line represents elution profile of theheterodimer—hIgG1.RR.K409R.Ab4.Ab4/hIgG1.EE.L368E.Ab4.Ab4.

FIGS. 23A-23C depict another example that ion exchange chromatographycan be used to separate IgG1 mutant (221R, 228R, and 409R) from IgG1(221E, 228E, and 368E) mutant even if the variable domains are the same.The heterodimer of these two variants can also be separated from thehomodimers using ion exchange chromatography. Dashed line represents Ab3antibody with 221R and 228R mutations in the hinge region and 409Rmutation in the CH3 domain of the heavy chain. Dotted line representsAb3 hIgG1 antibody with 221E and 228E mutations in the hinge and 368E inthe CH3 domain. Solid line represents elution profile of theheterodimer—hIgG1.RR.K409R.Ab3.Ab3/hIgG1.EE.L368E.Ab3.Ab3.

FIGS. 24A-24B depict (A) a comparison of bispecific antibody formationfor the indicated mutants (B).

FIGS. 25A-25B also depict (A) a comparison of bispecific antibodyformation for the indicated mutants (B).

FIGS. 26A-26D depict ion exchange elution profiles of hIgG1 and hIgG2mutants. Solid line represents elution profiles of the Ab2-Ab1bispecific antibody reaction products formed after incubation of the Ab2and Ab1 variants with gluthione (1 mM for hIgG1 and 2 mM for hIgG2). CH3only mutation provides about 12% IgG1 or 13% IgG2 heterodimeric proteinformation (mutations at K409R and L368E) in comparison to the wild-typehIgG1 and the combination of both the hinge (mutations at D221R, P228R,D221E, and P228E) and the CH3 mutations (mutations at K409R and L368E)provides about 90% IgG1 heterodimeric protein formation in comparison tothe wild-type hIgG1.

FIG. 27 depicts differential scanning calorimetry profiles for wild-typehIgG1 antibodies (Ab5 and Ab6), for parental mutant monospecificantibodies (hIgG1.RR.K409R.Ab6.Ab6/hIgG1.RR.K409R.Ab6.Ab6), and for thebispecific antibody ((hIgG1.EE.L368E.Ab5.Ab5/hIgG1.RR.K409R.Ab6.Ab6).

FIGS. 28A-28B depict sensorgrams showing the binding of a bispecificantibody to amine-coupled (antigen A)-hFc or (antigen D)-hFc and bindingto a panel of “sandwiching” analytes (or antigens).

FIG. 29 shows that growth inhibition by monospecific and bispecific Ab3and Ab4 was assayed in Cal27 (top panel) and FaDu (bottom panel) cells.Ab3.biFc (cross) is the parental mutant antibody(hIgG1.EE.L368E.Ab3.Ab3/hIgG1.EE.L368E.Ab3.Ab3). Ab3-Ab4 bispecificantibody (open square) is the bispecific mutant antibody(hIgG1.EE.L368E.Ab3.Ab3/hIgG1.RR.K409R.Ab4.Ab4). Ab3/nc.biFc (opencircle) is the monovalent Ab3 with a negative control antibody (Ab6) onone arm (hIgG1.RR.K409R.Ab3.Ab3/hIgG1.EE.L368E.Ab6.Ab6). Ab3.hIgG1(filled diamond) is the wild-type bivalent Ab3 in hIgG1 (Ab3.wild-typehIgG1). Ab4.hIgG1 (open triangle) is the wild-type bivalent Ab4 in hIgG1(Ab4.wild-type hIgG1). Ab4nc.biFc (open diamond) is the monovalent Ab4with a negative control antibody (Ab6) in one arm(hIgG1.RR.K409R.Ab4.Ab4/hIgG1.EE.L368E.Ab6.Ab6). “nc” denotes negativecontrol antibody.

FIG. 30 shows the dissociation rate constants of monovalent andbispecific Ab3 and Ab4 antibody measured in Cal27 cells. BispecificAb3/Ab4 antibody (black square) is the bispecific mutant antibody(hIgG1.EE.L368E.Ab3.Ab3/hIgG1.RR.K409R.Ab4.Ab4). Ab4/nc.biFc (cross) isthe monovalent Ab4 with a negative control (non-specific) antibody (Ab6)in one arm (hIgG1.RR.K409R.Ab4.Ab4/hIgG1.EE.L368E.Ab6.Ab6). Ab3/nc.biFc(open triangle) is the monovalent Ab3 with a negative control antibody(Ab6) on one arm (hIgG1.RR.K409R.Ab3.Ab3/hIgG1.EE.L368E.Ab6.Ab6). Solidand dotted lines are fit to a single exponential equation.

FIGS. 31A and 31B show that the ability of a heterodimeric protein(bispecific EpCAM/CD3 antibody ((labeled as “hG2-EpCAM-CD3” in thefigures)) to kill tumor cells (SW480) was mediated by cytotoxic T cellsin vitro. E/T denotes the ratio between the effector cells and thetarget cells.

DETAILED DESCRIPTION

The invention provides improved methods, compositions, kits and articlesof manufacture for generating heteromultimeric complex molecules, andmore particularly, heterodimeric proteins comprising at least oneimmunoglobulin-like hinge region, such as, e.g., a bispecific antibody.The invention provides methods to make and to purify heteromultimericcomplex molecules in pragmatic yields and desirable purities. Theinvention makes possible the efficient production of complex moleculesthat in turn can be used for diagnosing and/or treating variousdisorders or pathological conditions in which use of a molecule that ismultispecific in nature and highly stable is desirable and/or required.Details of methods, compositions, kits and articles of manufacture ofthe invention are provided herein.

General Techniques and Definitions

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclature used in connection with, and techniques of, cell and tissueculture, molecular biology, immunology, microbiology, genetics andprotein and nucleic acid chemistry and hybridization described hereinare those well known and commonly used in the art.

The methods and techniques of the present invention are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook J. & Russell D. Molecular Cloning: ALaboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2000); Ausubel et al., Short Protocols in MolecularBiology. A Compendium of Methods from Current Protocols in MolecularBiology, Wiley, John & Sons, Inc. (2002); Harlow and Lane UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1998); and Coligan et al., Short Protocols inProtein Science, Wiley, John & Sons, Inc. (2003). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications, as commonly accomplished in the art or as describedherein. The nomenclature used in connection with, and the laboratoryprocedures and techniques of, molecular biology, biochemistry,immunology, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are those wellknown and commonly used in the art. Throughout this specification andclaims, the word “comprise,” or variations such as “comprises” or“comprising,” will be understood to imply the inclusion of a statedinteger or group of integers but not the exclusion of any other integeror group of integers.

An “antibody” is an immunoglobulin molecule capable of specific bindingto a target, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc., through at least one antigen recognition site, located in thevariable region of the immunoglobulin molecule. As used herein, unlessotherwise indicated by context, the term is intended to encompass notonly intact polyclonal or monoclonal antibodies, but also fragmentsthereof (such as Fab, Fab′, F(ab′)₂, Fv), single chain (ScFv) and domainantibodies, including shark and camelid antibodies), and fusion proteinscomprising an antibody portion, multivalent antibodies, multispecificantibodies (e.g., bispecific antibodies so long as they exhibit thedesired biological activity) and antibody fragments as described herein,and any other modified configuration of the immunoglobulin molecule thatcomprises an antigen recognition site, for example without limitation,minibodies, maxibody, monobodies, peptibodies, intrabodies, diabodies,triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger andHudson, Nature Biotech. 23(9): 1126-1136 (2005)). An antibody includesan antibody of any class, such as IgG, IgA, or IgM (or sub-classthereof), and the antibody need not be of any particular class.Depending on the antibody amino acid sequence of the constant domain ofits heavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chainconstant domains that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion preferably retains at least one, preferably most orall, of the functions normally associated with that portion when presentin an intact antibody.

The residue designations in this application are based on the EUnumbering scheme of Kabat (Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, National Institutes of Health, Bethesda, Md.,ed. 5).

A “bivalent antibody” comprises two antigen binding sites per molecule(e.g., IgG). In some instances, the two binding sites have the sameantigen specificities. However, bivalent antibodies may be bispecific(see below).

A “monovalent antibody” comprises one antigen binding site per molecule(e.g., IgG). In some instances, a monovalent antibody can have more thanone antigen binding sites, but the binding sites are from differentantigens.

A “multispecific antibody” is one that targets more than one antigen orepitope. A “bispecific,” “dual-specific” or “bifunctional” antibody is ahybrid antibody having two different antigen binding sites. Bispecificantibodies are a species of multispecific antibody and may be producedby a variety of methods including, but not limited to, fusion ofhybridomas or linking of Fab′ fragments. See, e.g., Songsivilai &Lachmann (1990), Clin. Exp. Immunol. 79:315-321; and Kostelny et al.(1992), J. Immunol. 148:1547-1553. The two binding sites of a bispecificantibody will bind to two different epitopes, which may reside on thesame or different protein targets.

The phrase “antigen binding arm,” “target molecule binding arm,” andvariations thereof, as used herein, refers to a component part of anantibody of the invention that has an ability to specifically bind atarget molecule of interest. Generally and preferably, the antigenbinding arm is a complex of immunoglobulin polypeptide sequences, e.g.,CDR and/or variable domain sequences of an immunoglobulin light andheavy chain.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigen. Further, in contrast to polyclonal antibodypreparations that typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen.

The monoclonal antibodies herein may, in certain embodiments,specifically include “chimeric” antibodies in which a portion of theheavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, so long as they exhibit the desired biological activity(U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci.USA 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may, moreover, compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the followingreview articles and references cited therein: Vaswani and Hamilton, Ann.Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.5:428-433 (1994).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues.

As used herein, the term “immunoadhesin” designates antibody-like orimmunoglobulin-like molecules which combine the “binding domain” of aheterologous protein (an “adhesin”, e.g. a receptor, ligand or enzyme)with the effector component of immunoglobulin constant domains.Structurally, the immunoadhesins comprise a fusion of the adhesin aminoacid sequence with the desired binding specificity which is other thanthe antigen recognition and binding site (antigen combining site) of anantibody (i.e. is “heterologous”) and an immunoglobulin constant domainsequence. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG1,IgG2, IgG3, or IgG4 subtypes, IgA, IgE, IgD or IgM.

A “heteromultimer”, “heteromultimeric complex”, or “heteromultimericpolypeptide” is a molecule comprising at least a first polypeptide and asecond polypeptide, wherein the second polypeptide differs in amino acidsequence from the first polypeptide by at least one amino acid residue.The heteromultimer can comprise a “heterodimer” formed by the first andsecond polypeptide or can form higher order tertiary structures wherepolypeptides in addition to the first and second polypeptide arepresent.

A “heterodimer,” “heterodimeric protein,” “heterodimeric complex,” or“heteromultimeric polypeptide” is a molecule comprising a firstpolypeptide and a second polypeptide, wherein the second polypeptidediffers in amino acid sequence from the first polypeptide by at leastone amino acid residue.

The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” areused interchangeably herein to refer to chains of amino acids of anylength, preferably, relatively short (e.g., 10-100 amino acids). Thechain may be linear or branched, it may comprise modified amino acids,and/or may be interrupted by non-amino acids. The terms also encompassan amino acid chain that has been modified naturally or by intervention;for example, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation or modification,such as conjugation with a labeling component. Also included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),as well as other modifications known in the art. It is understood thatthe polypeptides can occur as single chains or associated chains.

The term “Fc region” as used herein generally refers to a dimer complexcomprising the C-terminal polypeptide sequences of an immunoglobulinheavy chain, wherein a C-terminal polypeptide sequence is that which isobtainable by papain digestion of an intact antibody. The Fc region maycomprise native or variant Fc sequences. The Fc sequence of animmunoglobulin generally comprises two constant domains, a CH2 domainand a CH3 domain, and optionally comprises a CH4 domain. The term “Fcpolypeptide” is used herein to refer to one of the polypeptides thatmakes up an Fc region. In some embodiments, an Fc polypeptide may beobtained or derived from any suitable immunoglobulin, such as from atleast one of the various IgG1, IgG2, IgG3, or IgG4 subtypes, or fromIgA, IgE, IgD or IgM. In some embodiments, an Fc polypeptide comprisespart or all of a wild-type hinge sequence (generally at its N terminus).In some embodiments, an Fc polypeptide does not comprise a wild-typehinge sequence. An Fc polypeptide may comprise native or variant Fcsequences.

By “Fc fusion” as used herein is meant a protein wherein one or morepolypeptides is operably linked to an Fc polypeptide. An Fc fusioncombines the Fc region of an immunoglobulin with a fusion partner, whichin general may be any protein, polypeptide or small molecule. Virtuallyany protein or small molecule may be linked to Fc to generate an Fcfusion. Protein fusion partners may include, but are not limited to, thetarget-binding region of a receptor, an adhesion molecule, a ligand, anenzyme, a cytokine, a chemokine, or some other protein or proteindomain. Small molecule fusion partners may include any therapeutic agentthat directs the Fc fusion to a therapeutic target. Such targets may beany molecule, for example without limitation, an extracellular receptorthat is implicated in disease.

The “hinge region,” “hinge sequence”, and variations thereof, as usedherein, includes the meaning known in the art, which is illustrated in,for example, Janeway et al., ImmunoBiology: the immune system in healthand disease, (Elsevier Science Ltd., NY) (4th ed., 1999); Bloom et al.,Protein Science (1997), 6:407-415; Humphreys et al., J. Immunol. Methods(1997), 209:193-202.

The “immunoglobulin-like hinge region,” “immunoglobulin-like hingesequence,” and variations thereof, as used herein, refer to the hingeregion and hinge sequence of an immunoglobulin-like or an antibody-likemolecule (e.g., immunoadhesins). In some embodiments, theimmunoglobulin-like hinge region can be from or derived from any IgG1,IgG2, IgG3, or IgG4 subtype, or from IgA, IgE, IgD or IgM, includingchimeric forms thereof, e.g., a chimeric IgG1/2 hinge region.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” (or simply, “recombinantvectors”). In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector.

“Polynucleotide,” or “nucleic acid molecule,” which may be usedinterchangeably herein, refers to a polymeric, possibly isolated, formof nucleosides or nucleotides of at least 10 bases in length. The termincludes single and double stranded forms. The nucleotides can bedeoxyribonucleotides, ribonucleotides, modified nucleotides or bases,and/or their analogs, or any substrate that can be incorporated into apolymer by DNA or RNA polymerase, or by a synthetic reaction.

A polynucleotide may comprise modified nucleotides, such as methylatednucleotides and their analogs. If present, modification to thenucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter synthesis, such as by conjugation with a label. Other types ofmodifications include, for example, “caps”, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotide(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid or semi-solidsupports. The 5′ and 3′ terminal OH can be phosphorylated or substitutedwith amines or organic capping group moieties of from 1 to 20 carbonatoms. Other hydroxyls may also be derivatized to standard protectinggroups. Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, T-O-methyl-, 2′-0-allyl, T-fluoro- or T-azido-ribose,carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars suchas arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs and abasic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), “(O)NR₂ (“amidate”), P(O)R,P(O)OR′, CO or CH₂ (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

“Oligonucleotide,” as used herein, generally refers to short, generallysingle stranded, generally synthetic polynucleotides that are generally,but not necessarily, less than about 200 nucleotides in length. Theterms “oligonucleotide” and “polynucleotide” are not mutually exclusive.The description above for polynucleotides is equally and fullyapplicable to oligonucleotides.

A reference to a nucleotide sequence as used herein encompasses itscomplement unless otherwise specified. Thus, a reference to a nucleicacid having a particular sequence should be understood to encompass itscomplementary strand, with its complementary sequence, unless otherwisedefined by context.

“Cell” or “cell line,” as used herein, includes various types of cellsthat can be used to express a heterodimeric protein, a polypeptide or anucleic acid of the invention, e.g., prokaryotic cells, eukaryoticcells, mammalian cells, rat cells, human cells.

The term “purify,” and grammatical variations thereof, is used to meanthe removal, whether completely or partially, of at least one impurityfrom a mixture containing the polypeptide and one or more impurities,which thereby improves the level of purity of the polypeptide in thecomposition (i.e., by decreasing the amount (ppm) of impurity(ies) inthe composition). According to the present invention, purification isperformed using at least one purification step that separates on thebasis of the electrostatic state of one or more of animmunoglobulin-like hinge polypeptide or region, and a CH3 region. Incertain embodiments, at least one purification step comprises orconsists essentially of ion-exchange chromatography.

The terms “ion-exchange” and “ion-exchange chromatography” refer to achromatographic process in which an ionizable solute of interest (e.g.,a protein of interest in a mixture) interacts with an oppositely chargedligand linked (e.g., by covalent attachment) to a solid phase ionexchange material under appropriate conditions of pH and conductivity,such that the solute of interest interacts non-specifically with thecharged compound more or less than the solute impurities or contaminantsin the mixture. The contaminating solutes in the mixture can be washedfrom a column of the ion exchange material or are bound to or excludedfrom the resin, faster or slower than the solute of interest.“Ion-exchange chromatography” specifically includes cation exchange,anion exchange, and mixed mode chromatographies.

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds. An “agonistantibody”, as used herein, is an antibody which mimics at least one ofthe functional activities of a polypeptide of interest.

The term “immune effector cell” or “effector cell as used herein refersto a cell within the natural repertoire of cells in the human immunesystem which can be activated to affect the viability of a target cell.The viability of a target cell can include cell survival, proliferation,and/or ability to interact with other cells.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.” Numeric ranges are inclusive of the numbers defining the range.

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, the present inventionencompasses not only the entire group listed as a whole, but each memberof the group individually and all possible subgroups of the main group,but also the main group absent one or more of the group members. Thepresent invention also envisages the explicit exclusion of one or moreof any of the group members in the claimed invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Exemplary methods and materialsare described herein, although methods and materials similar orequivalent to those described herein can also be used in the practice ortesting of the present invention. All publications and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. Although a number of documents are cited herein, this citationdoes not constitute an admission that any of these documents forms partof the common general knowledge in the art. Throughout thisspecification and claims, the word “comprise,” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer or group of integers but not the exclusion of any otherinteger or group of integers. Unless otherwise required by context,singular terms shall include pluralities and plural terms shall includethe singular. The materials, methods, and examples are illustrative onlyand not intended to be limiting.

Heteromultimeric Proteins

Except where indicated otherwise by context, the terms “first”polypeptide and “second” polypeptide, and variations thereof, are merelygeneric identifiers, and are not to be taken as identifying a specificor a particular polypeptide or component of heteromultimeric, e.g.heterodimeric proteins of the invention.

In one aspect, this invention provides a heteromultimeric protein, e.g.,a heterodimeric protein, comprising a hinge region, wherein the hingeregion comprises a first immunoglobulin-like hinge polypeptide and asecond immunoglobulin-like hinge polypeptide that interact together toform a hinge interface engineered to promote heterodimer formation,i.e., the first and second immunoglobulin-like hinge polypeptides tendto interact to form a heterodimeric hinge region faster and/or withgreater affinity or stability than do first or secondimmunoglobulin-like hinge polypeptides interact with like hinge (i.e.,first with first or second with second) polypeptides to form homodimerichinge regions. In certain embodiments of the invention, one or morecharged amino acids are present or are engineered within the hingeinterface so that they interact with one or more other amino acidswithin the hinge interface to electrostatically favor heterodimerformation over homodimer formation.

In some embodiments, the hinge region is an IgG, IgA, IgE, IgD, or IgMhinge region. In some embodiments, the hinge region is a human ornon-human mammal IgG region. In some embodiments, the hinge region is ahuman IgG1, IgG2, IgG3, or IgG4 hinge region, or chimeric versionsthereof.

In some embodiments or the heterodimeric protein of the invention, thefirst hinge polypeptide comprises one or more amino acid modificationrelative to a wild-type IgG hinge region, wherein the wild-type aminoacid is replaced with an amino acid having an opposite charge to thecorresponding amino acid in the second hinge polypeptide. In someembodiments or the heterodimeric protein of the invention, the firsthinge polypeptide comprises one or more amino acid modification relativeto a wild-type IgG hinge region; and the second hinge polypeptidecomprises at least one amino acid modification relative to a wild-typeIgG hinge region in proximity to, juxtaposed or at the same position asthe amino acid modification in the first hinge polypeptide, wherein thewild-type amino acid in the second hinge polypeptide is replaced with anamino acid having an opposite charge to the corresponding amino acid inthe first hinge polypeptide. As one of skill in the art will readilyappreciate, hinge polypeptides form three-dimensional structures andthus amino acids in the hinge region need not necessarily be identicalor contiguous in linear sequence in order to be in proximity to or tojuxtapose with one or more other amino acids in the hinge region inorder to “interact” in a non-covalent fashion, such as by electrostaticcharge.

In another aspect, this invention also provides a heterodimeric proteincomprising a hinge region and a CH3 region. In some embodiments, the CH3region is engineered to destabilize homodimer formation and promoteheterodimer formation. In some embodiments, the engineered CH3 region isnot electrostatically unfavorable to homodimer formation. In someembodiments, both the hinge region and the CH3 region are engineered toelectrostatically favor heterodimer formation over homodimer.

In another aspect, this invention also provides a heteromultimeric,e.g., a heterodimeric Fc fusion protein comprising a hinge region,wherein the hinge region comprises a first immunoglobulin hingepolypeptide and a second immunoglobulin hinge polypeptide that meetinteract together to form a hinge interface engineered to promoteheterodimeric Fc fusion protein formation, wherein one or more chargedamino acids within the hinge interface electrostatically promoteheterodimeric Fc fusion protein formation. Examples of heterodimeric Fcfusion proteins include, without limitation, bispecific antibodies,monospecific antibodies, and multispecific antibodies. In someembodiments, the heterodimeric Fc fusion protein is an antibody. Inother embodiments, the heterodimeric Fc fusion protein is not anantibody.

In another aspect, this invention also provides a bispecific antibodycomprising a hinge region, wherein the hinge region comprises a firstimmunoglobulin hinge polypeptide and a second immunoglobulin hingepolypeptide that meet interact together to form a hinge interfaceengineered to promote bispecific antibody formation, wherein one or morecharged amino acids within the hinge interface electrostatically promotebispecific antibody formation.

In another aspect, this invention also provides a bispecific antibody orFc fusion heterodimeric protein comprising a hinge region and a CH3region, wherein the hinge region and/or the CH3 region are engineered tofavor heterodimer formation over homodimer. In some embodiments, thehinge region is engineered to electrostatically favor heterodimerformation over homodimer. In some embodiments, the engineered CH3 regionis not electrostatically unfavorable to homodimer formation.

In another aspect, this invention also provides a bispecific antibody orFc fusion heterodimeric protein comprising a hinge region and a CH3region, wherein the CH3 region is engineered to favor heterodimerformation over homodimer. In some embodiments, the engineered CH3 regionis not electrostatically unfavorable to homodimer formation.

In some embodiments, a heterodimeric protein of the invention maycomprise two antibody fragments, such as, for example, an Fc or Fcfusion polypeptide. An Fc fusion polypeptide generally comprises an Fcsequence (or fragment thereof) fused to a heterologous polypeptidesequence, such as, for example without limitation, an antigen bindingdomain. One exemplary Fc fusion polypeptide is a receptor extracellulardomain (ECD) fused to an immunoglobulin Fc sequence (e.g., Fit receptorECD fused to an IgG2 Fc).

In certain embodiments, the amino acid modification(s) in a hinge region(for example without limitation, a human IgG4, IgG2 or IgG1 hingeregion) occur(s) at any one or more residues of the hinge region. Insome embodiments, the amino acid modification(s) occur(s) at one or morethe following positions of a hinge region: 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229 and 230.

In certain embodiments, the amino acid modification(s) in a CH3 region(for example without limitation, a human IgG4, IgG2 or IgG1 CH3 region)occur(s) at any one or more residues of the CH3 region. In someembodiments, the amino acid modification(s) occur(s) at one or more thefollowing positions of a CH3 region: 341, 342, 343, 344, 345, 346, 347,348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361,362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375,376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389,390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403,404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417,418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431,432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445,446 and 447.

In some embodiments, the amino acid modification(s) in the hinge region(e.g., human IgG1 hinge region) occur(s) at a position selected from219, 221, 227 and 228. In some embodiments, the amino acidmodification(s) in the hinge region (e.g., human IgG1 hinge region)occur(s) at a position selected from 221 and 228. In some embodiments,the amino acid modification(s) in the CH3 region (e.g., human IgG1 CH3region) occur(s) at a position selected from 349, 368, 405, and 409. Insome embodiments, the amino acid modification(s) in the CH3 regionincludes K409R, L368D, and/or L368E. In some embodiments, the amino acidmodifications occur at positions 221 and 228 (e.g., (D221R or D221E) and(P228R or P228E)) in the hinge region and at position 409 or 368 (e.g.,K409R or L368E) in the CH3 region of human IgG 1.

In some embodiments, the amino acid modification in the hinge region(e.g., human IgG2 hinge region) is located at a position selected from222, 223, 225, 227 and 228. In some embodiments, the amino acidmodification in the hinge region (e.g., human IgG2 hinge region) islocated at a position selected from 223, 225 and 228. In someembodiments, the amino acid modification in the CH3 region (e.g., humanIgG2 CH3 region) is located at a position selected from 349, 368, 405,and 409. In some embodiments, the amino acid modification in the CH3region (e.g., human IgG2 CH3 region) is located at a position selectedfrom 368 and 409. In some embodiments, the amino acid modification(s) inthe CH3 region includes K409R, L368D, and/or L368E. In some embodiments,the amino acid modifications occur at positions 223, 225, and 228 (e.g.,(C223E or C223R), (E225E or E225R), and (P228E or P228R)) in the hingeregion and at position 409 or 368 (e.g., K409R or L368E) in the CH3region of human IgG2.

In some embodiments, the amino acid modification in the hinge region(e.g., human IgG4 hinge region) is located at a position selected from217 and 228. In some embodiments, the amino acid modification(s) in thehinge region includes a modification at position 228.

In another aspect, the invention also provides a polypeptide comprisinga hinge region engineered to electrostatically favor heterodimer overhomodimer formation. In some embodiments, a polypeptide of the inventioncomprises a heavy chain constant domain and a light chain constantdomain. In some embodiments, a polypeptide of the invention comprisesone or more modified amino acids in the Fc region (e.g., hinge region orhinge and CH3 region), which is capable of electrostatically promotingheterodimer formation. In one embodiment, a polypeptide of the inventiondoes not comprise a modification in the CH3 region. In some embodiments,a portion (but not all) of the Fc sequence is missing in an antibody ofthe invention. In some of these embodiments, the missing Fc sequence isat least a portion of, in some case the entire, CH2 and/or CH3 domain.

In some embodiments, a heterodimeric hinge and/or CH3 region comprise(s)any of the substitution combinations shown in rows 1-67 Table 1. In someembodiments, a first hinge and/or CH3 polypeptide comprise(s) any of thesubstitution combinations shown in Table 1. In some embodiments, asecond hinge and/or CH3 polypeptide comprise(s) any of the substitutioncombinations shown in Table 1. In Table 1, positions are shown in bold(i.e., hinge positions 221, 223, 225 and 228 and CH3 positions 368 and409). Rows 1-15 correspond to substitutions made in an IgG1 hinge and/orIgG2 CH3. Rows 16-63 correspond to substitutions made in an IgG2 hingeand/or CH3. Rows 64-67 correspond to substitutions made in an IgG4hinge. E/D indicates a substitution at position 368 in a CH3 polypeptidewith either Glu or Asp.

TABLE 1 Substitutions in first hinge-CH3 polypeptide Substitutions insecond hinge-CH3 polypeptide 221 223 225 228 368 409 221 223 225 228 368409 1. R R E E 2. R E E R 3. R R R E E R 4. R E R E R R 5. R R E/D E EE/D 6. R E E/D E R E/D 7. R R E/D E E R 8. R E E/D E R R 9. R R R E E E10. R E R E R E 11. R R E E E R 12. R E E E R R 13. R R 14. E/D E/D 15.E/D R 16. D E K R R D 17. D E K R R R D R 18. D E K R R R D E/D 19. D EK E/D R R D R 20. D E K R R D 21. D E E R K R 22. D E E R R K R R 23. DE E R R K R E/D 24. D E E E/D R K R R 25. D E E E/D R K R E/D 26. E R ER E K 27. E R E R R E K R 28. E R E R R E K E/D 29. E R E E/D R E K R30. E R E E/D R E K E/D 31. E E R R K E 32. E E R R R K E R 33. E E R RR K E E/D 34. E E R E/D R K E R 35. E E R E/D R K E E/D 36. D R E K E R37. D R E R K E R R 38. D R E R K E R E/D 39. D R E E/D K E R R 40. D RE E/D K E R E/D 41. D E R R R E 42. D E R R R R E R 43. D E R R R R EE/D 44. D E R E/D R R E R 45. D E R E/D R R E E/D 46. E E E R R R 47. EE E R R R R R 48. E E E R R R R E/D 49. E E E E/D R R R R 50. E E E E/DR R R E/D 51. E K E R E R 52. E K E R R E R R 53. E K E R R E R E/D 54.E K E E/D R E R R 55. E K E E/D R E R E/D 56. D E E R K R 57. D E E R RK R R 58. D E E R R K R E/D 59. D E E E/D R K R R 60. D E E E/D R K RE/D 61. R R 62. E/D E/D 63. E/D R 64. R E 65. K E 66. R D 67. K D

In another aspect of the invention, the heterodimeric protein (e.g.,bispecific antibody) as described herein comprises a full-length humanantibody, wherein a first antibody variable domain of the heterodimericprotein is capable of recruiting the activity of a human immune effectorcell by specifically binding to an effector antigen located on the humanimmune effector cell, and wherein a second antibody variable domain ofthe heterodimeric protein is capable of specifically binding to a targetantigen. In some embodiments, the human antibody has an IgG1, IgG2,IgG3, or IgG4 isotype. In some embodiments, the heterodimeric proteincomprises an immunologically inert Fc region.

The human immune effector cell can be any of a variety of immuneeffector cells known in the art. For example, the immune effector cellcan be a member of the human lymphoid cell lineage, including, but notlimited to, a T cell (e.g., a cytotoxic T cell), a B cell, and a naturalkiller (NK) cell. The immune effector cell can also be, for examplewithout limitation, a member of the human myeloid lineage, including,but not limited to, a monocyte, a neutrophilic granulocyte, and adendritic cell. Such immune effector cells may have either a cytotoxicor an apoptotic effect on a target cell or other desired effect uponactivation by binding of an effector antigen.

The effector antigen is an antigen (e.g., a protein or a polypeptide)that is expressed on the human immune effector cell. Examples ofeffector antigens that can be bound by the heterodimeric protein (e.g.,a heterodimeric protein or a bispecific antibody) include, but are notlimited to, human CD3 (or CD3 (Cluster of Differentiation) complex),CD16, NKG2D, NKp46, CD2, CD28, CD25, CD64, and CD89.

The target cell can be a cell that is native or foreign to humans. In anative target cell, the cell may have been transformed to be a malignantcell or pathologically modified (e.g., a native target cell infectedwith a virus, a plasmodium, or a bacterium). In a foreign target cell,the cell is an invading pathogen, such as a bacterium, a plasmodium, ora virus.

The target antigen is expressed on a target cell in a diseased condition(e.g., an inflammatory disease, a proliferative disease (e.g., cancer),an immunological disorder, a neurological disease, a neurodegenerativedisease, an autoimmune disease, an infectious disease (e.g., a viralinfection or a parasitic infection), an allergic reaction, agraft-versus-host disease or a host-versus-graft disease). A targetantigen is not effector antigen. Examples of the target antigensinclude, but are not limited to, EpCAM (Epithelial Cell AdhesionMolecule), CCR5 (Chemokine Receptor type 5), CD19, HER (Human EpidermalGrowth Factor Receptor)-2/neu, HER-3, HER-4, EGFR (Epidermal GrowthFactor Receptor), PSMA, CEA, MUC-1 (Mucin), MUC2, MUC3, MUC4, MUC5AC,MUC5B, MUC7, CIhCG, Lewis-Y, CD20, CD33, CD30, ganglioside GD3,9-O-Acetyl-GD3, GM2, Globo H, fucosyl GM1, Poly SA, GD2, CarboanhydraseIX (MN/CA IX), CD44v6, Shh (Sonic Hedgehog), Wue-1, Plasma Cell Antigen,(membrane-bound) IgE, MCSP (Melanoma Chondroitin Sulfate Proteoglycan),CCR8, TNF-alpha precursor, STEAP, mesothelin, A33 Antigen, PSCA(Prostate Stem Cell Antigen), Ly-6; desmoglein 4, E-cadherin neoepitope,Fetal Acetylcholine Receptor, CD25, CA19-9 marker, CA-125 marker and MIS(Muellerian Inhibitory Substance) Receptor type II, sTn (sialylated Tnantigen; TAG-72), FAP (fibroblast activation antigen), endosialin,EGFRvIII, LG, SAS and CD63.

In some embodiments, the heterodimeric protein (e.g., bispecificantibody) as described herein comprises a full-length human antibody,wherein a first antibody variable domain of the heterodimeric protein iscapable of recruiting the activity of a human immune effector cell byspecifically binding to an effector antigen (e.g., CD3 antigen) locatedon the human immune effector cell, wherein a second antibody variabledomain of the heterodimeric protein is capable of specifically bindingto a target antigen (e.g., CD20 antigen or EpCAM), wherein both thefirst and the second antibody variable domains of the heterodimericprotein comprise amino acid modifications at positions 221 and 228(e.g., (D221R or D221E) and (P228R or P228E)) in the hinge region and atposition 409 or 368 (e.g., K409R or L368E) in the CH3 region.

In some embodiments, the heterodimeric protein (e.g., bispecificantibody) as described herein comprises a full-length human antibody,wherein a first antibody variable domain of the heterodimeric protein iscapable of recruiting the activity of a human immune effector cell byspecifically binding to an effector antigen (e.g., CD3 antigen) locatedon the human immune effector cell, wherein a second antibody variabledomain of the heterodimeric protein is capable of specifically bindingto a target antigen (e.g., CD20 antigen or EpCAM), wherein both thefirst and the second antibody variable domains of the heterodimericprotein comprise amino acid modifications at positions 223, 225, and 228(e.g., (C223E or C223R), (E225E or E225R) and (P228E or P228R)) in thehinge region and at position 409 or 368 (e.g., K409R or L368E) in theCH3 region.

In another aspect of the invention, a heterodimeric protein disclosedherein may be deimmunized to reduce immunogenicity upon administrationto a subject using known techniques such as those described, e.g., inPCT Publication WO98/52976 and WO00/34317.

In other embodiments, a heterodimeric Fc fusion protein may be modifiedor derivatized, such as by making a fusion antibody or immunoadhesinthat comprises all or a portion of the heterodimeric polypeptide, e.g.,bispecific antibody disclosed herein, linked to another polypeptide ormolecular agent. Heteromultimeric, e.g. heterodimeric polypeptidesdisclosed herein (e.g., bispecific antibodies) may be modified orderivatized, for example, to extend in vivo half-lives, by producingmore stable fusion molecules and/or by treatment with biocompatiblepolymers such as polyethylene glycol (PEG), commonly referred to as“pegylation,” or by any of a number of other engineering methods wellknown in the art.

A heterodimeric Fc fusion protein may be derivatized with a chemicalgroup, including but not limited to polyethylene glycol (PEG), a methylor ethyl group, an ester, a carbohydrate group and the like, using wellknown techniques. These chemical groups (and others like them which havebeen used to stability therapeutic compounds in vivo) are useful toimprove the biological characteristics of the heterodimeric polypeptide,e.g., to increase serum half-life and bioactivity.

A heterodimeric Fc fusion protein may also be labeled using any of amultitude of methods known in the art. As used herein, the terms “label”or “labeled” refers to incorporation of another molecule in theantibody. In one embodiment, the label is a detectable marker, e.g.,incorporation of a radiolabeled amino acid or attachment to apolypeptide of biotinyl moieties that can be detected by marked avidin(e.g., streptavidin containing a fluorescent marker or enzymaticactivity that can be detected by optical or colorimetric methods). Inanother embodiment, the label or marker can be therapeutic, e.g., a drugconjugate or toxin. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to: radioisotopes orradionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I),fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors),enzymatic labels (e.g., horseradish peroxidase, β-galactosidase,luciferase, alkaline phosphatase), chemiluminescent markers, biotinylgroups, predetermined polypeptide epitopes recognized by a secondaryreporter (e.g., leucine zipper pair sequences, binding sites forsecondary antibodies, metal binding domains, epitope tags), magneticagents, such as gadolinium chelates, toxins such as pertussis toxin,taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. In some embodiments, labels are attached by spacerarms of various lengths to reduce potential steric hindrance.

Nucleic Acids, Vectors and Cells

The present invention also encompasses nucleic acid molecules andsequences encoding polypeptides disclosed herein that comprise modifiedimmunoglobulin-like hinge or Fc related sequences. In some embodiments,different nucleic acid molecules encode one or more chains or portionsof the heterodimeric protein, e.g., bispecific antibody disclosedherein. In other embodiments, the same nucleic acid molecule encodes aheterodimeric protein disclosed herein.

In one aspect, the present invention provides a nucleic acid sequenceencoding one of the chains of a heterodimeric protein disclosed herein,or portion thereof as described above. Nucleic acid molecules of theinvention include nucleic acids that hybridize under highly stringentconditions, such as those at least about 70%, 75%, 80%, 85%, 90%, 95%,97%, 98% or 99% or more identical to a nucleic acid sequence of theinvention.

The term “percent sequence identity” in the context of nucleic acidsequences means the residues in two sequences that are the same whenaligned for maximum correspondence. The length of sequence identitycomparison may be over a stretch of at least about nine nucleotides,usually at least about 18 nucleotides, more usually at least about 24nucleotides, typically at least about 28 nucleotides, more typically atleast about 32 nucleotides, and preferably at least about 36, 48 or morenucleotides. There are a number of different algorithms known in the artwhich can be used to measure nucleotide sequence identity. For instance,polynucleotide sequences can be compared using FASTA, Gap or Bestfit,which are programs in Wisconsin Package Version 10.0, Genetics ComputerGroup (GCG), Madison, Wis. FASTA, which includes, e.g., the programsFASTA2 and FASTA3, provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol.132:185-219 (2000); Pearson, Methods Enzymol. 266:227-258 (1996);Pearson, J. Mol. Biol. 276:71-84 (1998); incorporated herein byreference). Unless otherwise specified, default parameters for aparticular program or algorithm are used. For instance, percent sequenceidentity between nucleic acid sequences can be determined using FASTAwith its default parameters (a word size of 6 and the NOPAM factor forthe scoring matrix) or using Gap with its default parameters as providedin GCG Version 6.1, incorporated herein by reference.

In a further aspect, the present invention provides a vector comprisinga nucleic acid sequence encoding one or more of the chains or portionsof the heteromultimeric or heterodimeric protein disclosed herein, orportion thereof as described herein.

In a further aspect, the present invention provides a vector suitablefor expressing one or more of the chains or portions of theheterodimeric protein disclosed herein, or portion thereof as describedherein.

In another embodiment, a nucleic acid molecule of the invention is usedas a probe or PCR primer for a specific amino acid sequence, e.g., aspecific antibody sequence such as in hinge and constant heavy domainsequences. For instance, the nucleic acid can be used as a probe indiagnostic methods or as a PCR primer to amplify regions of DNA thatcould be used, inter alia, to isolate additional nucleic acid moleculesencoding useful sequences. In some embodiments, the nucleic acidmolecules are oligonucleotides. In some embodiments, theoligonucleotides are from hinge and constant domain regions of the heavyand light chains of an antibody of interest. In some embodiments, theoligonucleotides encode all or a part of one or more of the modifiedhinge regions of the heterodimeric polypeptide, e.g., bispecificantibodies or fragments thereof of the invention as described herein.

Recombinant expression vectors of the invention may, in someembodiments, carry regulatory sequences that control the expression ofantibody chain genes in a host cell. It will be appreciated by thoseskilled in the art that the design of the expression vector, includingthe selection of regulatory sequences may depend on such factors as thechoice of the host cell to be transformed, the level of expression ofprotein desired, etc. Preferred regulatory sequences for mammalian hostcell expression include viral elements that direct high levels ofprotein expression in mammalian cells, such as promoters and/orenhancers derived from retroviral LTRs, cytomegalovirus (CMV) (such asthe CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40promoter/enhancer), adenovirus, (e.g., the adenovirus major latepromoter (AdMLP)), polyoma and strong mammalian promoters such as nativeimmunoglobulin and actin promoters. For further description of viralregulatory elements, and sequences thereof, see e.g., U.S. Pat. Nos.5,168,062, 4,510,245 and 4,968,615. Methods for expressing antibodies inplants, including a description of promoters and vectors, as well astransformation of plants is known in the art. See, e.g. U.S. Pat. No.6,517,529. Methods of expressing polypeptides in bacterial cells orfungal cells, e.g., yeast cells, are also well known in the art.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017). For example, typically theselectable marker gene confers resistance to drugs, such as G418,hygromycin or methotrexate, on a host cell into which the vector hasbeen introduced. For example, selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in dhfr⁻ host cells withmethotrexate selection/amplification), the neo gene (for G418selection), and the glutamate synthetase gene.

The term “expression control sequence” as used herein meanspolynucleotide sequences that are necessary to effect the expression andprocessing of coding sequences to which they are ligated. Expressioncontrol sequences include appropriate transcription initiation,termination, promoter and enhancer sequences; efficient RNA processingsignals such as splicing and polyadenylation signals; sequences thatstabilize cytoplasmic mRNA; sequences that enhance translationefficiency (i.e., Kozak consensus sequence); sequences that enhanceprotein stability; and when desired, sequences that enhance proteinsecretion. The nature of such control sequences differs depending uponthe host organism; in prokaryotes, such control sequences generallyinclude promoter, ribosomal binding site, and transcription terminationsequence; in eukaryotes, generally, such control sequences includepromoters and transcription termination sequence. The term “controlsequences” is intended to include, at a minimum, all components whosepresence is essential for expression and processing, and can alsoinclude additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences.

Methods of Producing Heteromultimeric Proteins

In one aspect, this invention provides a strategy for enhancing theformation of a desired heteromultimeric or heterodimeric protein, e.g.,an Fc fusion protein, by altering or engineering an interface between afirst and a second immunoglobulin-like Fc region (e.g., a hinge region,a CH3 region, or a hinge region and a CH3 region). In some embodiments,one or more residues that make up the hinge interface are replaced withcharged residues such that the electrostatic interactions between thesecharged residues electrostatically favor heterodimer formation overhomodimer formation. In further embodiments, one or more residues thatmake up the CH3 interface are further replaced with charged residuessuch that the interactions between the CH3 interface further promotesheterodimer formation over homodimer formation. In some embodiments, theengineered CH3 interface electrostatically favor heterodimer formationover homodimer formation. In some embodiments, the engineered CH3interface sterically favor heterodimer formation over homodimerformation. In other embodiments, the engineered CH3 interfacedestabilizes homodimer formation but is not electrostaticallyunfavorable to homodimer formation.

In some embodiments, the formation of the heterodimeric proteincomprising one or more amino acid modification in the first hinge regionand the first CH3 region disclosed herein is substantially increased incomparison to the wild-type heterodimeric protein without suchmodifications. In some embodiments, the formation of the heterodimericprotein comprising one or more amino acid modification in the firsthinge region and the first CH3 region is at least about any of 51%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% in comparisonto the wild-type heterodimeric protein without such modifications. Insome embodiments, the amino acid modification(s) in the hinge regionoccur(s) at a position selected from 217, 218, 219, 221, 222, 223, 224,225, 226, 227, and 228. In some embodiments, the amino acidmodification(s) in the CH3 region occur(s) at a position selected from349, 368, 405, and 409.

In some embodiments, the formation of the heterodimeric proteincomprising one or more amino acid modification in both the first andsecond hinge regions and both the first and second CH3 regions disclosedherein is substantially increased in comparison to the wild-typeheterodimeric protein without such modifications. In some embodiments,the formation of the heterodimeric protein comprising one or more aminoacid modification in both the first and second hinge regions and boththe first and second CH3 regions is at least about any of 51%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% in comparison tothe wild-type heterodimeric protein without such modifications. In someembodiments, the amino acid modification(s) in the hinge region occur(s)at a position selected from 217, 218, 219, 221, 222, 223, 224, 225, 226,227, and 228. In some embodiments, the amino acid modification(s) in theCH3 region occur(s) at a position selected from 349, 368, 405, and 409.

In another aspect, this invention also provides methods of producing aheteromultimeric protein, e.g., a heterodimeric protein of theinvention.

In some embodiments, the method comprising the steps of: a) culturing ahost cell comprising a nucleic acid molecule encoding a firstpolypeptide comprising a modified Fc region (e.g., hinge region and/orCH3 region) and the same or a different nucleic acid molecule encoding asecond polypeptide comprising a modified Fc region (e.g., hinge regionand/or CH3 region), wherein the cultured host cell expresses the firstand second polypeptides; and b) recovering the heteromultimeric, e.g.,heterodimeric protein from the host cell culture. In some embodiments,the first polypeptide and second polypeptides are two different Fcfusion polypeptides. In some embodiments, the first polypeptide andsecond polypeptides are two different antibody heavy chains. In someembodiments, the host cell further expresses another polypeptide, e.g.,a light chain. In some embodiments, the light chain can associate withboth heavy chains. Methods of coexpressing two different heavy chainswith a single light chain are described in detail in, e.g., Example 3below.

In some embodiments, the method comprising the steps of: a) expressingthe first polypeptide in a first host cell; b) expressing the secondpolypeptide in a second host cell; c) isolating the first polypeptide ofstep a) and the second polypeptide of step b); and d) incubating the twopolypeptides of step c) and the isolated polypeptide of step c) under acondition suitable for multimer formation, e.g., dimerization, toproduce the heteromultimeric, e.g., heterodimeric protein. In someembodiments, the molecules or antibodies may be mixed in a salinesolution containing a suitable reducing agent (e.g., glutathione). Anysuitable saline solution and appropriate pH may be used, e.g., one thatcomprises Dulbecco's phosphate buffered saline (D-PBS). In someembodiments, the first and/or second host cell further expresses anotherpolypeptide, e.g., a light chain.

The skilled artisan can readily determine, using well-known techniques,the relative amounts of molecules or antibodies to use according to themethods disclosed herein.

In the methods disclosed herein, incubations may be performed across arange of temperatures. Such temperatures will be recognized by thoseskilled in the art and will include, for example, incubationtemperatures at which deleterious physical changes such as denaturationor decomposition do not occur in the mixed molecules or antibodies. Incertain embodiments, the incubations are performed at 37° C.

Any of a number of host cells may be used in methods of the invention.Such cells are known in the art (some of which are described herein) orcan be determined empirically with respect to suitability for use inmethods of the invention using routine techniques known in the art. Incertain embodiments, the host cell is prokaryotic. In some embodiments,a host cell is a gram-negative bacterial cell. In other embodiments, ahost cell is E. coli. In some embodiments, the E. coli is of a straindeficient in endogenous protease activities. In some embodiments, thegenotype of an E. coli host cell lacks degP and prc genes and harbors amutant spr gene. In other embodiments of the invention, the host cell ismammalian, for example, a Chinese Hamster Ovary (CHO) cell.

In some embodiments, methods of the invention further compriseexpressing in a host cell a polynucleotide or recombinant vectorencoding a molecule the expression of which in the host cell enhancesyield of a bispecific antibody or a heterodimeric protein of theinvention. For example, such molecule can be a chaperone protein. In oneembodiment, said molecule is a prokaryotic polypeptide selected from thegroup consisting of DsbA, DsbC, DsbG and FkpA. In some embodiments ofthese methods, the polynucleotide encodes both DsbA and DsbC.

Non-Hybridoma Host Cells and Methods of Recombinantly Producing Protein

In one aspect, the present invention provides recombinant host cellsallowing the recombinant expression of the antibodies of the inventionor portions thereof. Antibodies produced by such recombinant expressionin such recombinant host cells are referred to herein as “recombinantantibodies”. The present invention also provides progeny cells of suchhost cells, and antibodies produced by same. The term “recombinant hostcell” (or simply “host cell”), as used herein, means a cell into which arecombinant expression vector has been introduced. It should beunderstood that “recombinant host cell” and “host cell” mean not onlythe particular subject cell but also the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein. Such cell may comprise a vectoraccording to the invention as described above.

In another aspect, the present invention provides a method for making anantibody or portion thereof as described above. According to oneembodiment, said method comprises culturing a cell transfected ortransformed with a vector as described above, and retrieving saidantibody or portion thereof. Nucleic acid molecules encoding antibodiesand vectors comprising these nucleic acid molecules can be used fortransfection of a suitable mammalian, plant, bacterial or yeast hostcell. Transformation can be by any known method for introducingpolynucleotides into a host cell. Methods for introduction ofheterologous polynucleotides into mammalian cells are well known in theart and include dextran-mediated transfection, calcium phosphateprecipitation, polybrene-mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,and direct microinjection of the DNA into nuclei. In addition, nucleicacid molecules may be introduced into mammalian cells by viral vectors.Methods of transforming cells are well known in the art. See, e.g., U.S.Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455. Methods oftransforming plant cells are well known in the art, including, e.g.,Agrobacterium-mediated transformation, biolistic transformation, directinjection, electroporation and viral transformation. Methods oftransforming bacterial and yeast cells are also well known in the art.

Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC). These include, inter alia,Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells,293 Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, babyhamster kidney (BHK) cells, African green monkey kidney cells (COS),human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and anumber of other cell lines. Cell lines of particular preference areselected through determining which cell lines have high expressionlevels. Other cell lines that may be used are insect cell lines, such as519 or Sf21 cells. When recombinant expression vectors encoding antibodygenes are introduced into mammalian host cells, the antibodies areproduced by culturing the host cells for a period of time sufficient toallow for expression of the antibody in the host cells or, morepreferably, secretion of the antibody into the culture medium in whichthe host cells are grown. Antibodies can be recovered from the culturemedium using standard protein purification methods. Suitable plant hostcells may include, e.g., Nicotiana, Arabidopsis, duckweed, corn, wheat,potato, etc. Suitable bacterial host cells may include, e.g., E. coliand Streptomyces species. Suitable yeast host cells may include, e.g.,Schizosaccharomyces pombe, Saccharomyces cerevisiae and Pichia pastoris.

Expression of polypeptides of the invention or portions thereof fromproduction cell lines can be enhanced using a number of knowntechniques. For example, the glutamine synthetase gene expression system(the GS system) is a common approach for enhancing expression undercertain conditions. The GS system is discussed in whole or part inconnection with EP patents 0 216 846, 0 256 055, 0 323 997 and 0 338841.

It is likely that polypeptides comprising Fc polypeptides or Fc regionsand immunoglobulin-like hinge polypeptides, such as, e.g., antibodies,as expressed by different cell lines or in transgenic animals, willdiffer from each other in their glycosylation patterns. All such“glycoforms” of polypeptides of the invention, including allheterodimers of polypeptides comprising immunoglobulin-like hingesequences, bispecific polypeptides, antibodies and the like, areconsidered to be part of the instant invention, regardless of theirglycosylation state, and more generally, regardless of the presence orabsence of any post-translational modification(s).

Methods of Purifying Heteromultimeric Proteins

In another aspect, the invention provides a method of purifyingheterodimeric proteins on the basis of the electrostatic state (e.g.,electric charge difference) of one or more of an immunoglobulin-likehinge polypeptide or region, and/or a CH3 region by chromatography.Disclosed herein are chromatographic methods of isolating heterodimericproteins from a mixture comprising heterodimeric proteins andhomodimeric proteins on the basis of the electrostatic state (e.g.,electric charge difference) of one or more of an immunoglobulin-likehinge polypeptide or region, and/or a CH3 region. The electrostaticstate or electric charge differences can be influenced by ionic strengthand/or pH level.

Chromatographies can include, for example, affinity chromatography, ionexchange chromatography, hydrophobic interaction chromatography,hydroxyapatite chromatography, gel filtration chromatography,reverse-phase chromatography, and adsorption chromatography. Liquidphase chromatography (e.g., HPLC (High-Performance (or Pressure) LiquidChromatography) and FPLC (Fast Protein Liquid Chromatography)) can beused for carrying out the chromatographies disclosed above. Examples ofcolumns for affinity chromatography include protein A (synthetic,recombinant, or native) columns and protein G (synthetic, recombinant,or native) columns.

In some embodiments, the purified heterodimeric protein preparationresulting from chromatography is highly pure, i.e., having less thanabout any of 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.1, 0.1, 0.01 percent to nohomodimer present. In some embodiments, the chromatography is ionexchange chromatography.

In some embodiments, the heterodimeric protein to be purified comprisesan immunoglobulin-like Fc region, wherein the Fc region comprises afirst Fc polypeptide and a second Fc polypeptide which interact togetherto form an Fc interface, wherein electrostatic interactions between oneor more charged amino acids within the Fc interface favor heterodimerformation over homodimer formation, wherein the purification isperformed using at least one purification step that separates on thebasis of the electrostatic state of one or more of animmunoglobulin-like hinge polypeptide or region, and a CH3 region. Incertain embodiments, at least one purification step comprises orconsists essentially of a step of ion exchange chromatography method. Insome embodiments, purification step consists of a step of ion exchangechromatography method. Any other suitable methods for purifying aheterodimeric protein comprising an immunoglobulin-like Fc region,wherein the Fc region comprises a first Fc polypeptide and a second Fcpolypeptide which interact together to form a Fc interface, whereinelectrostatic interactions between one or more charged amino acidswithin the Fc interface favor heterodimer formation over homodimerformation may be used to purify heteromultimeric proteins, e.g.,heterodimers disclosed herein and are encompassed by the presentinvention.

In some embodiments, the heterodimeric protein to be purified comprisesan immunoglobulin-like hinge region, wherein the hinge region comprisesa first hinge polypeptide and a second hinge polypeptide which interacttogether to form a hinge interface, wherein electrostatic interactionsbetween one or more charged amino acids within the hinge interface favorheterodimer formation over homodimer formation, wherein the purificationcomprises or consists essentially of a step of ion exchangechromatography method of purifying a heterodimeric protein comprising animmunoglobulin-like hinge region, wherein the hinge region comprises afirst hinge polypeptide and a second hinge polypeptide which interacttogether to form a hinge interface, wherein electrostatic interactionsbetween one or more charged amino acids within the hinge interface favorheterodimer formation over homodimer formation. In some embodiments,purification step consists of a step of ion exchange chromatographymethod.

In some embodiments, the heterodimeric protein to be purified comprisesan immunoglobulin-like CH3 region, wherein the CH3 region comprises afirst CH3 polypeptide and a second CH3 polypeptide which interacttogether to form a CH3 interface, wherein electrostatic interactionsbetween one or more charged amino acids within the CH3 interface favorheterodimer formation over homodimer formation, wherein the purificationcomprises or consists essentially of a step of ion exchangechromatography method of purifying a heterodimeric protein comprising animmunoglobulin-like CH3 region, wherein the CH3 region comprises a firstCH3 polypeptide and a second CH3 polypeptide which interact together toform a CH3 interface, wherein electrostatic interactions between one ormore charged amino acids within the CH3 interface favor heterodimerformation over homodimer formation. In some embodiments, purificationstep consists of a step of ion exchange chromatography method.

Methods of Using Heteromultimeric Proteins

The present invention also provides various therapeutic applications forthe heteromultimeric proteins (e.g., heterodimeric polypeptide orbispecific antibody) as described herein. In one aspect, theheteromultimeric proteins can be used for treating various diseases(e.g., cancer, autoimmune diseases, or viral infections) by binding thefirst protein (e.g., first human antibody variable domain) to aneffector antigen and by binding the second protein (e.g., second humanantibody variable domain) to a target antigen. For example, theheteromultimeric proteins (e.g., heterodimeric polypeptide or bispecificantibody) can be used for redirecting cytotoxicity, deliveringthrombolytic agents to clots, for delivering immunotoxins to tumorcells, or for converting enzyme activated prodrugs at a target site(e.g., a tumor).

In another aspect, the heteromultimeric proteins (e.g., heterodimericpolypeptide or bispecific antibody) can be used for increasingspecificity of a therapeutic agent and/or modulating synergistic oradditive pathways (e.g., metabolic or biochemical pathways). Forexample, the heteromultimeric proteins (e.g., heterodimeric polypeptideor bispecific antibody) can engage receptor/receptor, receptor/ligand,ligand/ligand, cell/cell, ligand/payload, receptor/payload, or singlereceptor.

Pharmaceutical Compositions

In one aspect, the present invention provides a pharmaceuticalcomposition comprising a heteromultimeric, e.g., heterodimericpolypeptide, e.g., bispecific antibody, of the invention or portionthereof as described above in a pharmaceutically acceptable carrier. Incertain embodiments, the polypeptides of the invention may be present ina neutral form (including zwitter ionic forms) or as a positively ornegatively-charged species. In some embodiments, the polypeptides may becomplexed with a counterion to form a “pharmaceutically acceptablesalt,” which refers to a complex comprising one or more polypeptides andone or more counterions, where the counterions are derived frompharmaceutically acceptable inorganic and organic acids and bases.

The heterodimeric proteins, or portions thereof, may be administeredalone or in combination with one or more other polypeptides of theinvention or in combination with one or more other drugs (or as anycombination thereof). The pharmaceutical compositions, methods and usesof the invention thus also encompass embodiments of combinations(co-administration) with other active agents, as detailed below.

As used herein, the terms “co-administration,” “co-administered” and “incombination with,” referring to the antibodies of the invention and oneor more other therapeutic agents, is intended to mean, and does refer toand include the following: (i) simultaneous administration of suchcombination of a heterodimer disclosed herein and therapeutic agent(s)to a patient in need of treatment, when such components are formulatedtogether into a single dosage form which releases said components atsubstantially the same time to said patient; (ii) substantiallysimultaneous administration of such combination of a heterodimerdisclosed herein and therapeutic agent(s) to a patient in need oftreatment, when such components are formulated apart from each otherinto separate dosage forms which are taken at substantially the sametime by said patient, whereupon said components are released atsubstantially the same time to said patient; (iii) sequentialadministration of such combination of a heterodimer disclosed herein andtherapeutic agent(s) to a patient in need of treatment, when suchcomponents are formulated apart from each other into separate dosageforms which are taken at consecutive times by said patient with asignificant time interval between each administration, whereupon saidcomponents are released at substantially different times to saidpatient; and (iv) sequential administration of such combination of aheterodimer disclosed herein and therapeutic agent(s) to a patient inneed of treatment, when such components are formulated together into asingle dosage form which releases said components in a controlled mannerwhereupon they are concurrently, consecutively, and/or overlappinglyreleased at the same and/or different times to said patient, where eachpart may be administered by either the same or a different route.

Generally, the heterodimeric proteins disclosed herein or portionsthereof are suitable to be administered as a formulation in associationwith one or more pharmaceutically acceptable excipient(s). The term‘excipient’ is used herein to describe any ingredient other than thecompound(s) of the invention. The choice of excipient(s) will to a largeextent depend on factors such as the particular mode of administration,the effect of the excipient on solubility and stability, and the natureof the dosage form. As used herein, “pharmaceutically acceptableexcipient” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. Some examplesof pharmaceutically acceptable excipients are water, saline, phosphatebuffered saline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Additional examples ofpharmaceutically acceptable substances are wetting agents or minoramounts of auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the antibody.

Pharmaceutical compositions of the present invention and methods fortheir preparation will be readily apparent to those skilled in the art.Such compositions and methods for their preparation may be found, forexample, in Remington's Pharmaceutical Sciences, 19th Edition (MackPublishing Company, 1995). Pharmaceutical compositions are preferablymanufactured under GMP conditions.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage. Any method foradministering peptides, proteins or antibodies accepted in the art maysuitably be employed for the heterodimeric proteins and portions thereofdisclosed herein.

The pharmaceutical compositions of the invention are typically suitablefor parenteral administration. As used herein, “parenteraladministration” of a pharmaceutical composition includes any route ofadministration characterized by physical breaching of a tissue of asubject and administration of the pharmaceutical composition through thebreach in the tissue, thus generally resulting in the directadministration into the blood stream, into muscle, or into an internalorgan. Parenteral administration thus includes, but is not limited to,administration of a pharmaceutical composition by injection of thecomposition, by application of the composition through a surgicalincision, by application of the composition through a tissue-penetratingnon-surgical wound, and the like. In particular, parenteraladministration is contemplated to include, but is not limited to,subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous,intraarterial, intrathecal, intraventricular, intraurethral,intracranial, intrasynovial injection or infusions; and kidney dialyticinfusion techniques. Preferred embodiments include the intravenous andthe subcutaneous routes.

Formulations of a pharmaceutical composition suitable for parenteraladministration typically generally comprise the active ingredientcombined with a pharmaceutically acceptable carrier, such as sterilewater or sterile isotonic saline. Such formulations may be prepared,packaged, or sold in a form suitable for bolus administration or forcontinuous administration. Injectable formulations may be prepared,packaged, or sold in unit dosage form, such as in ampoules or in multidose containers containing a preservative. Formulations for parenteraladministration include, but are not limited to, suspensions, solutions,emulsions in oily or aqueous vehicles, pastes, and the like. Suchformulations may further comprise one or more additional ingredientsincluding, but not limited to, suspending, stabilizing, or dispersingagents. In one embodiment of a formulation for parenteraladministration, the active ingredient is provided in dry (i.e. powder orgranular) form for reconstitution with a suitable vehicle (e.g. sterilepyrogen free water) prior to parenteral administration of thereconstituted composition. Parenteral formulations also include aqueoussolutions which may contain excipients such as salts, carbohydrates andbuffering agents (preferably to a pH of from 3 to 9), but, for someapplications, they may be more suitably formulated as a sterilenon-aqueous solution or as a dried form to be used in conjunction with asuitable vehicle such as sterile, pyrogen-free water. Exemplaryparenteral administration forms include solutions or suspensions insterile aqueous solutions, for example, aqueous propylene glycol ordextrose solutions. Such dosage forms can be suitably buffered, ifdesired. Other parentally-administrable formulations which are usefulinclude those which comprise the active ingredient in microcrystallineform, or in a liposomal preparation. Formulations for parenteraladministration may be formulated to be immediate and/or modifiedrelease. Modified release formulations include controlled, delayed,sustained, pulsed, targeted and programmed release formulations. Forexample, in one aspect, sterile injectable solutions can be prepared byincorporating the heterodimeric protein, e.g., bispecific antibody, inthe required amount in an appropriate solvent with one or a combinationof ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile filteredsolution thereof. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

An exemplary, non-limiting pharmaceutical composition of the inventionis a formulation as a sterile aqueous solution having a pH that rangesfrom about 5.0 to about 6.5 and comprising from about 1 mg/mL to about200 mg/mL of a heterodimeric protein disclosed herein, from about 1millimolar to about 100 millimolar of histidine buffer, from about 0.01mg/mL to about 10 mg/mL of polysorbate 80, from about 100 millimolar toabout 400 millimolar of trehalose, and from about 0.01 millimolar toabout 1.0 millimolar of disodium EDTA dihydrate.

Dosage regimens may be adjusted to provide the optimum desired response.For example, a single bolus may be administered, several divided dosesmay be administered over time or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation. It is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form, as used herein, refers tophysically discrete units suited as unitary dosages for thepatients/subjects to be treated; each unit containing a predeterminedquantity of active compound calculated to produce the desiredtherapeutic effect in association with the required pharmaceuticalcarrier. The specification for the dosage unit forms of the inventionare generally dictated by and directly dependent on (a) the uniquecharacteristics of the chemotherapeutic agent and the particulartherapeutic or prophylactic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

Thus, the skilled artisan would appreciate, based upon the disclosureprovided herein, that the dose and dosing regimen is adjusted inaccordance with methods well-known in the therapeutic arts. That is, themaximum tolerable dose can be readily established, and the effectiveamount providing a detectable therapeutic benefit to a patient may alsobe determined, as can the temporal requirements for administering eachagent to provide a detectable therapeutic benefit to the patient.Accordingly, while certain dose and administration regimens areexemplified herein, these examples in no way limit the dose andadministration regimen that may be provided to a patient in practicingthe present invention.

It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated, and may include single or multipledoses. It is to be further understood that for any particular subject,specific dosage regimens should be adjusted over time according to theindividual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.Further, the dosage regimen with the compositions of this invention maybe based on a variety of factors, including the type of disease, theage, weight, sex, medical condition of the patient, the severity of thecondition, the route of administration, and the particular antibodyemployed. Thus, the dosage regimen can vary widely, but can bedetermined routinely using standard methods. For example, doses may beadjusted based on pharmacokinetic or pharmacodynamic parameters, whichmay include clinical effects such as toxic effects and/or laboratoryvalues. Thus, the present invention encompasses intra-patientdose-escalation as determined by the skilled artisan. Determiningappropriate dosages and regimens are well-known in the relevant art andwould be understood to be encompassed by the skilled artisan onceprovided the teachings disclosed herein.

For administration to human subjects, the total monthly dose of aheterodimeric protein disclosed herein is typically in the range ofabout 0.5 to about 1200 mg per patient, depending, of course, on themode of administration. For example, an intravenous monthly dose mayrequire about 1 to about 1000 mg/patient. The total monthly dose may beadministered in single or divided doses and may, at the physician'sdiscretion, fall outside of the typical range given herein.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of a heterodimeric protein, e.g., abispecific antibody or portion thereof, disclosed herein is about 1 toabout 1000 mg/patient/month. In certain embodiments, the heterodimericprotein may be administered at about 1 to about 200 or about 1 to about150 mg/patient/month.

EXAMPLES

The following examples describe construction, generation, andpurification of heterodimeric proteins comprising mutations in the hingeregion only, in both the hinge region and CH3 regions, or in the CH3region only. The following examples are meant to illustrate the methodsand materials of the present invention. Suitable modifications andadaptations of the described conditions and parameters normallyencountered in the art that are obvious to those skilled in the art arewithin the spirit and scope of the present invention.

Antibodies Used in the Examples

Antibody Identifier Antibody Description* Ab1 anti-antigen A antibodyAb2 anti-antigen B antibody comprising a lambda light chain Ab3anti-antigen C antibody Ab4 anti-antigen D antibody Ab5 Non-antigenbinding antibody Ab6 Non-antigen binding antibody *Antigen A is ahormone protein; antigens B and C are two different growth factorreceptor proteins; and antigen D is a calcium signal transducer protein.

Example 1: Generation of Human IgG1, IgG2, and IgG4 Antibody MutantClones

PCR Mutagenesis

In this and other Examples below, the mutant clones of human IgG1, IgG2,and IgG4 antibodies were generated by PCR mutagenesis. For human IgG2antibody mutant clones, an anti-antigen A antibody (also referred asAb1) having IgG2ΔA Fc region (SEQ ID NO: 1 in FIG. 2) was used as thetemplate (about 0.05 μg per reaction) for two steps of PCR reactions(FIG. 4). Compared to a wild-type IgG2 Fc region, this IgG2ΔA has A330Sand P331S substitutions. For all the PCR reaction described in thisexample, the PfuTurbo® DNA Polymerase Kit (catalog number 600250) wasused and the final dNTP concentration was 0.5 mM.

In the first step, there were two separate PCR reactions—A and B. Inreaction A, a first pair of primers, hFc2.f (forward primer; SEQ ID NO:2 in Table 2) and hFc2.hinge.r (reverse primer; SEQ ID NO: 3 in Table2), was used at 40 pmol each. In reaction B, a second pair of primers,hFc2.hinge.mutA1.f (forward primer which contains mutations; SEQ ID NO:4 in Table 2) and Not.hFc2.r (reverse primer; SEQ ID NO: 5 in Table 2)was used at 40 pmol each. The forward primer—hFc2. hinge.mutA1.fcomprised mutated nucleic acids compared to a wild-type IgG2 hingeregion and introduced the desired mutations into the hinge region. Theanneal temperature for PCR reactions described here is 54° C. Theprimers used in the PCR reactions comprise degenerate nucleotides, i.e.,“S” in the sequence stands for C or G in the primer and “R” stands for Aor G in the primer.

The PCR products obtained from reaction A and reaction B were gelpurified by QIAquick Gel Exaction Kit (catalog number 28706) and furthereluted in 30 μl EB buffer.

In the second step of PCR reactions, 2.5 μl of the purified PCR productsfrom reaction A and reaction B, respectively, were amplified for 8cycles first without the addition of any primers and then were subjectedto 20 cycles with the following primers (40 pmol each)—forward primer:hFc2.f (SEQ ID NO: 2) and reverse primer: Not.hFc2.r (SEQ ID NO: 5). Theanneal temperature for PCR reactions described here is 54° C.

The PCR products obtained from the second step were gel purified byQIAquick Gel Exaction Kit (catalog number 28706) and further eluted in30 μl EB buffer. The purified PCR products were digested by ApaI andNotI and further cloned into either a pCi.db.3×FLAG.Ab1.hFc1 vector or apCi.db.HA.Ab1.hFc1 vector (FIG. 3).

A number of different mutation-containing primers (SEQ ID NOS: 6-10)were used to replace hFc2.hinge.mutA1.f in the 1st step PCR reaction tointroduce different mutations into the hinge region. A human IgG2ΔAantibody mutant—IgG2ΔA (K409) having a K409R substitution in the CH3region was also used as a template to replace the human IgG2ΔA antibodytemplate to generate different mutations in the hinge region.

The IgG1 and IgG4 antibody mutants were generated by a PCR mutagenesisprocess, using essentially the same as procedures as previouslydescribed. For human IgG1 antibody mutant clones, DNA encoding antibodyAb1 IgG1 (SEQ ID NO: 11 in FIG. 4) having a K409R substitution in theCH3 region was used as the PCR template. For human IgG4 antibody mutantclones, a wild-type antibody Ab1 IgG4 Fc region (SEQ ID NO: 12 in FIG.5) was used as the PCR template. The primers used for generation of IgG1and IgG4 mutant clones are listed in Table 2.

Mutant Clones

Multiple mutant clones were generated for human IgG1, IgG2 and IgG4antibodies with mutated residues in the hinge regions. For human IgG4antibody mutant clones, the residue Ser228 in a wild-type Fc hingeregion (see the underlined residue in FIG. 6A) was mutated to either apositively charged residue (Lys or Arg; group A) or to a negativelycharged residue (Asp or Glu; group B) in the mutant clones listed in thetable of FIG. 6B. For human IgG2 antibody mutant clones in groups AI andBI, the human IgG2ΔA antibody having A330S and P331S substitutionscompared to IgG2 was used as the template. For mutant clones in groupsAI and BI, the human IgG2ΔA antibody mutant—IgG2ΔA (K409) having a K409Rsubstitution in the CH3 region, was used as the template. The threeresidues Cys223, Glu225, and Pro228 in the hinge region of the antibodytemplate, as underlined in FIG. 6A, were mutated, respectively, toeither a positively charged residue (Arg or Lys) or a negatively chargedresidue (Glu or Asp) to produce the mutant clones listed in the table ofFIG. 6B. For human IgG1 antibody mutant clones, as described above, thehuman IgG1 (K409R) mutant was used as the template and the residuesSer221 and Pro228 in the hinge region, as underlined in FIG. 6A, wererespectively mutated to either a positive charged residue (Arg or Lys)or a negative charged residue (Glu or Asp) to generate the mutant clonesas listed in the table of FIG. 6B.

To distinguish mutants having different Fc regions, the group A mutantsof IgG4 and the groups AI and AII mutants of IgG2 were engineered tofurther comprise an N-terminus 3×FLAG tag (DYKDHDGDYKDHDIDYKDDDDKGLE,SEQ ID NO: 53), while group B mutants of IgG4 and groups BI and BIImutants of IgG2 were engineered to further comprise an N-terminus HA tag(YPYDVPDYALE, SEQ ID NO: 54).

TABLE 2 PCR Primer Sequences for Generating Hinge  Region MutationsSEQ ID Primer Name Primer Sequence NO hFc2.f GCCTCCACCAAGGGCCCATC SEQ IDNO: 2 Not.hFc2.r ATACAAGCGGCCGCCTATTTACCCGG SEQ ID AGACAGGGA NO: 5hFc2.hinge. ACAAGACCGTGGAGAGAAAGTGTGA SEQ ID mutA1.fSGTGGAGTGTCCAARGTGTCCAGCCC NO: 4 CTCCAGTGG hFc2.hinge.ACAAGACCGTGGAGAGAAAGTGTGA SEQ ID mutA2.f SGTGGAGTGTCCAGASTGTCCAGCCCNO: 6 CTCCAGTGG hFc2.hinge. ACAAGACCGTGGAGAGAAAGTGTGA SEQ ID mutA3.fSGTGARGTGTCCAGASTGTCCAGCCC NO: 7 CTCCAGTGG hFc2.hinge.ACAAGACCGTGGAGAGAAAGTGTAR SEQ ID mutB1.f GGTGARGTGTCCAGASTGTCCAGCCCNO: 8 CTCCAGTGG hFc2.hinge. ACAAGACCGTGGAGAGAAAGTGTAR SEQ ID mutB2.fGGTGARGTGTCCAARGTGTCCAGCCC NO: 9 CTCCAGTGG hFc2.hinge.ACAAGACCGTGGAGAGAAAGTGTAR SEQ ID mutB3.f GGTGGAGTGTCCAARGTGTCCAGCCNO: 10 CCTCCAGTGG hFc2.hinge.r CTTTCTCTCCACGGTCTTG SEQ ID NO: 3 hFc4.fGCCTCCACCAAGGGCCCATC SEQ ID NO: 13 Not.hFc4.r ATACAAGCGGCCGCCTATTTACCCAGSEQ ID AGACAGGGAGA NO: 14 hFc4.hinge. GAGTCCAAATATGGTCCCCCATGCCC SEQ IDmutA.f AARGTGCCCAGCACCTGAGTTCCT NO: 15 hFc4.hinge.GAGTCCAAATATGGTCCCCCATGCCC SEQ ID mutB.f AGASTGCCCAGCACCTGAGTTCCT NO: 16hFc4.hinge. TGGGGGACCATATTTGGACT SEQ ID  mut.r NO: 17 hFc1.fGCCTCCACCAAGGGCCCATC SEQ ID NO: 18 Not.hFc1.r ATACAAGCGGCCGCCTATTTACCCGGSEQ ID AGACAGGGA NO: 19 hFc1.hinge. GAAAGTTGAGCCCAAATCTTGTGAG SEQ IDEE.f AAAACTCACACATGCCCAGAGTGCC NO: 20 CAGCACCTGAACTCC hFc1.hinge.GAAAGTTGAGCCCAAATCTTGTAGG SEQ ID RR.f AAAACTCACACATGCCCAAGGTGCC NO: 21CAGCACCTGAACTCC hFc1.hinge.r ACAAGATTTGGGCTCAACTTTC SEQ ID NO: 22

Example 2: IgG4 Hinge-Containing Heterodimers

This Example illustrates heterodimeric proteins containing mutant IgG4hinge.

The human IgG4 antibody group A and group B mutants were mixed togetherin four different combination pairs (as shown in FIG. 7B). Each pair wasco-transfected with antibody Ab1 light chain into 293F cells grown insuspension culture. Briefly, 293F cells were seeded at 1×106 cells/ml in293 Freestyle media in Erlenmeyer flasks (8% CO2, 120 rpm). For thetransfection (amounts based on a 50 ml transfection, can be scaled up asneeded), 2.5 ml of OptiMEM was first added to 2× 15 ml tubes. 50 μg ofDNA (heavy chain A:heavy chain B:light chain=1.5:1.5:2) was then addedto tube A. 100 μl of a 1 mg/ml solution of transfection reagent wasadded to tube B. The materials in tubes A and B were mixed together andincubated at RT for 15 minutes. A DNA-transfection reagent complexsolution was added to cells and then the cells were returned toincubator. After 24 hours, 1.25 ml of a 20% w/v stock of Tryptone N1 wasadded and the cells were returned to incubator. Supernatants wereharvested after 5 days. The transfection reagent was prepared bydissolving it to 1 mg/ml in water, adjusting pH to below 2.5 with HCl.After dissolving, the pH was further adjusted to 7.0 followed by 0.22 μmfiltration (aliquot and store at −20° C.). The Tryptone N1 was made a20% w/v stock in 293Freestyle media, and followed by 0.22 μm filtration(store at 4° C.).

Total proteins in each preparation were separated using immunoaffinitypurification on a protein G column (Protein G agarose, Pierce cat#20399; IgG elution buffer, Pierce cat #21004; See, e.g., Bjorck andKronvall, J. Immunol. (133): 969-974 (1984)). The percentage ofbispecific antibodies in each preparation were measured by a standardsandwich ELISA assay. Briefly, plates were coated with anti-HA and thedetection antibody was anti-FLAG. The antibody Ab1 having a wild-typeIgG4 region was expressed and purified in the same manner and used as astandard control for the ELISA assays because wild-type IgG4 naturallyforms about 50% bispecific antibody (van der Neut Kolfschoten M et al.,Science (317): 1554-1557 (2007); Aalberse R C et al., Immunology(105):9-19 (2002)).

To detect the bispecific antibodies, 0.1 μg/ml purified total proteinfrom each preparation was added into each ELISA plate with 1 μg/ml ofanti-HA tag antibody. The bispecific antibodies in each preparation weredetected by reacting with an HRP-conjugated anti-FLAG antibody.

The ELISA assay results (FIG. 7A) demonstrate that introducing one ormore mutations to drive heterodimer formation based on favorableelectrostatic interactions between hinge regions of heterodimerscompared to homodimers in a human IgG4 hinge region helped stabilizeheterodimeric antibody formation and thus produced more bispecificantibodies than the same procedure using only a wild-type IgG4 antibody.

Example 3: IgG2 Hinge-Containing Heterodimers

The human IgG2 antibody group AI mutants and group BI mutants were mixedtogether in three different combination pairs, 1A, 1B, and 1C (as shownin FIG. 8B). Each pair was co-transfected with antibody Ab1 light chaininto 293 cells. The human IgG2 antibody group AII mutants and group BIImutants were also mixed together in three different combination pairs,2A, 2B, and 2C (as shown in FIG. 8B). Each pair was co-transfected withantibody Ab1 light chain into suspension 293 cells. Supernatants wereharvested after 5 days. Total proteins in each preparation were purifiedby protein G column. The percentage of bispecific antibodies in eachpreparation were measured by sandwich ELISA. The antibody Ab1 withwild-type IgG2 Fc region was expressed and purified in the same mannerand used as a standard control for the ELISA assay. As described inExample 1, all the mutants in 1A, 1B, and 1C have a wild-type IgG2ΔA CH3region, and all the mutants in 2A, 2B, and 2C have K409R mutation in theCH3 region of IgG2ΔA.

To detect the bispecific antibodies, 0.1 μg/ml purified total proteinfrom each preparation was added into ELISA plate with 1 μg/ml anti-HAtag antibody. The bispecific antibodies in each preparation weredetected by HRP-conjugated anti-FLAG antibody.

The ELISA assay results (FIG. 8A) demonstrate that introducing the K409mutation in the CH3 region of human IgG2ΔA antibody helped promoteheterodimeric antibody formation.

Example 4: Screening for the IgG2 Hinge Mutation that PromotesHeterodimer Formation in K409R Background

The human IgG2 antibody group AII mutants and group BII mutants werecombined in six different combination pairs, A-F (as shown in FIG. 9B).Each pair of clones was co-transfected with antibody Ab1 light chaininto 293 cells. Supernatants were harvested after 5 days. Total proteinsin each preparation were purified by protein G column. The percentage ofbispecific antibodies in each preparation were measured by sandwichELISA. The antibody Ab1 with wild-type IgG2 Fc region and with wild-typeIgG4 Fc region were individually expressed and purified in the samemanner and used both controls for the ELISA assay. All the mutant clonesused in this Example have K409R mutation in the CH3 region of IgG2ΔA.

To detect the bispecific antibodies, 0.17 μg/ml purified total proteinfrom each preparation was added into ELISA plate coated with 1 μg/mlanti-HA tag antibody. The bispecific antibodies in each preparation weredetected by HRP-conjugated anti-FLAG antibody.

The ELISA assay results (FIG. 9A) demonstrate that in the K409Rbackground, when three hinge mutations—C223E, E225E, and P228E, combinedwith three hinge mutations C223R, E225R, and P228R, i.e., column D inFIG. 9A, we observed more bispecific antibodies than the other mutationcombinations tested.

Example 5: “Glu” Scanning of Human IgG4 CH3 Regions

Fourteen positions from the CH3 regions of the human IgG4 antibody werechosen to carry out a series of “Glu” scanning experiment. Criteria forchoosing these fourteen positions were essentially as described in W.Dall'Acqua et al. Biochemistry (37):9266-9273 (1998). The positionschosen for the “Glu” scanning were numbered 1-14 as shown in FIG. 10.All the mutants were generated using a site-directed mutagenesis kitfrom Stratagene (QuikChange® II XL Site-Directed Mutagenesis Kit,Catalog #200522). The primers used for generating specific mutation inthe CH3 region are listed in Table 3.

The template clone used to generate the IgG4 mutants was Ab1.3.11A,which has an N-terminal 3×FLAG tag and a S228R mutation in its hingeregion.

All the mutant clones and the template clones, as listed in FIG. 11B,were expressed and purified individually. Equal amounts of Ab1.3.2Aprotein, which has a S228E mutation in the hinge region and a N-terminalHA tag, and various Ab1.3.11A CH3 mutants were mixed together in variouscombinations (FIG. 11B) and further incubated with 0.5 mM glutathione(GSH) at 37° C. for 24 hour. The Ab1.3.11A template without any CH3region mutation was also mixed with equal amounts of Ab1.3.2A proteinand further incubated with 0.5 mM glutathione (GSH) at 37° C. for 24hour (column 15 in FIG. 11A). The protocol for the GSH reaction wasessentially as described in Labrijn et al., Nature Biotechnology, (27),767-771 (2009).

TABLE 3 PCR Primer Sequences for Generating CH3 Region Mutations SEQ IDPrimer Name hFc1 Primer Sequence NO Fc1.Q347E.fCCCCGAGAACCAGAGGTGTACACCCTG SEQ ID NO: 23 Fc1.Q347E.rCAGGGTGTACACCTCTGGTTCTCGGGG SEQ ID NO: 24 Fc1.Y349E.fGAGAACCACAGGTGGAGACCCTGCCCCCAT SEQ ID NO: 25 Fc1.Y349E.rATGGGGGCAGGGTCTCCACCTGTGGTTCTC SEQ ID NO: 26 Fc1.T350E.fAACCACAGGTGTACGAGCTGCCCCCATCCC SEQ ID NO: 27 Fc1.T350E.rGGGATGGGGGCAGCTCGTACACCTGTGGTT SEQ ID NO: 28 Fc1.L351E.fCACAGGTGTACACCGAGCCCCCATCCCGGG SEQ ID NO: 29 Fc1.L351E.rCCCGGGATGGGGGCTCGGTGTACACCTGTG SEQ ID NO: 30 Fc1.T366E.fCCAGGTCAGCCTGGAGTGCCTGGTCAAAGG SEQ ID NO: 31 Fc1.T366E.rCCTTTGACCAGGCACTCCAGGCTGACCTGG SEQ ID NO: 32 Fc1.L368E.fCAGCCTGACCTGCGAGGTCAAAGGCTTCTA SEQ ID NO: 33 Fc1.L368E.rTAGAAGCCTTTGACCTCGCAGGTCAGGCTG SEQ ID NO: 34 Fc1.K370E.fTGACCTGCCTGGTCGAGGGCTTCTATCCCA SEQ ID NO: 35 Fc1.K370E.rTGGGATAGAAGCCCTCGACCAGGCAGGTCA SEQ ID NO: 36 Fc1.K392E.fGGAGAACAACTACGAGACCACGCCTCCCGT SEQ ID NO: 37 Fc1.K392E.rACGGGAGGCGTGGTCTCGTAGTTGTTCTCC SEQ ID NO: 38 Fc1.T394E.fCAACTACAAGACCGAGCCTCCCGTGCTGGA SEQ ID NO: 39 Fc1.T394E.rTCCAGCACGGGAGGCTCGGTCTTGTAGTTG SEQ ID NO: 40 Fc1.V397E.fGACCACGCCTCCCGAGCTGGACTCCGACGG SEQ ID NO: 41 Fc1.V397E.rCCGTCGGAGTCCAGCTCGGGAGGCGTGGTC SEQ ID NO: 42 Fc1.L398E.fACGCCTCCCGTGGAGGACTCCGACGGCTCC SEQ ID NO: 43 Fc1.L398E.rGGAGCCGTCGGAGTCCTCCACGGGAGGCGT SEQ ID NO: 44 Fc1.F405E.fGACGGCTCCTTCGAGCTGTACAGCAAGCTC SEQ ID NO: 45 Fc1.F405E.rGAGCTTGCTGTACAGCTCGAAGGAGCCGTC SEQ ID NO: 46 Fc1.Y407E.fCTCCTTCTTCCTCGAGAGCAAGCTCACCG SEQ ID NO: 47 Fc1.Y407E.rCGGTGAGCTTGCTCTCGAGGAAGAAGGAG SEQ ID NO: 48 Fc1.K409E.fTTCCTCTACAGCGAGCTGACCGTGGACAAGA SEQ ID NO: 49 Fc1.K409E.rTCTTGTCCACGGTCAGCTCGCTGTAGAGGAA SEQ ID NO: 50 Fc1.K409R.fTTCCTCTACAGCAGGCTGACCGTGGACAAGA SEQ ID NO: 51 Fc1.K409R.rTCTTGTCCACGGTCAGCCTGCTGTAGAGGAA SEQ ID NO: 52

The aliquot from each GSH reaction was diluted in ice-cold PBS-TB (PBSwith 0.2% BSA, 0.05% Tween-20) and the amount of bispecific antibodieswas measured by sandwich ELISA as described in Example 2. The antibodyAb1 with a wild-type IgG4 Fc region was also expressed and treated inthe same manner and used as a standard control (column 16 in FIG. 11A)in the ELISA assay.

As shown in FIG. 11A, none of the fourteen mutations in IgG4 CH3 regionmade a significant increase in bispecific antibody formation compared tothe template clone without any CH3 region mutation.

Example 6: “Glu” Scanning of Human IgG2 CH3 Regions

Fourteen (14) positions from the CH3 regions of the human IgG2 antibodywere chosen to carry out a series of “Glu” scanning experiment. Criteriafor choosing these fourteen positions were essentially as described inExample 5. The positions chosen for the “Glu” scanning were numbered1-14 as shown in FIG. 10. All the mutants were generated by site-directmutagenesis kit from Stratagene.

The template used to generate the IgG2 mutants was Ab1.1.3D, which hasan N-terminal HA tag, a wild-type IgG2ΔA CH3 region, and threemutations, i.e., C223R, E225R, and P228R, in the IgG2ΔA hinge region.

All the mutants and the wild-type controls, as listed in FIG. 12B, wereexpressed and purified individually. Equal amounts of the Ab1.2.2Hprotein, which has three mutations in the hinge region, C223E, E225E,and P228E and an N-terminal HA tag, and various Ab1.1.3D CH3 mutants(FLAG tag) were mixed together in various combination (FIG. 12B) andfurther incubated with 0.5 mM GSH at 37° C. for 24 hours. The aliquotfrom each GSH reaction was diluted in ice-cold PBS-TB (PBS with 0.2%BSA, 0.05% Tween-20) and the amount of bispecific antibodies formed wasmeasured by sandwich ELISA as described in Example 2. The antibody Ab1with a wild-type IgG4 Fc region, the antibody Ab1 with a wild-typeIgG2ΔA Fc region and the antibody Ab1 with a mutant IgG2ΔA (K409R) Fcregion were expressed and treated in the same manner and used ascontrols in the ELISA assays.

As shown in FIG. 12A, when mixing the Ab1.2.2H protein (threemutations—C223E, E225E, and P228E) with the Ab1.1.3D.L368E protein(three mutations—C223R, E225R, and P228R), the production of bispecificantibodies (column 6 in FIG. 12A) was significantly increased comparedto the other combinations and the controls.

Example 7: Hinge Mutations and CH3 Mutations can Contribute toHeterodimer Formation

The Ab1 heavy chain variable region of some mutants was replaced withthe heavy chain variable region from a different antibody, Ab2, which isan anti-antigen B antibody comprising a lambda light chain.

All the mutants and the wild-type controls listed in FIG. 13B wereexpressed and purified individually. Equal amounts of antibody 1 aslisted in FIG. 13B and antibody 2 as listed in FIG. 13B were mixedtogether and incubated with or without 0.5 mM GSH at 37° C. for 24 hour.Aliquots from each GSH reaction were diluted in ice-cold PBS-TB (PBSwith 0.2% BSA, 0.05% Tween-20) and the amount of bispecific antibodieswas measured by sandwich ELISA, essentially as described in Example 2.

To detect bispecific antibodies, 0.25 μg/ml purified total proteins fromeach GSH reaction were added onto an ELISA plate coated with 1 μg/mlantigen B. The amount of bispecific antibodies in each preparation wasdetected by HRP conjugated anti-kappa antibody. Antibody Ab1 is ananti-antigen A antibody comprising a kappa light chain.

As shown in FIG. 13A, when two different wild-type human IgG2ΔAantibodies were mixed together under mild reducing condition (A) (1 mMGSH), no bispecific antibodies were detected compared to the controls.Similar to the results from Example 4, in the K409R background, whenthree hinge mutations: C223E, E225E, and P228E, combined with threehinge mutations C223R, E225R, and P228R, i.e., (B) in FIG. 13A,increased formation of bispecific antibodies was observed. Similar tothe results from Example 7, replacement of the K409R mutation with L368Eon one of the heavy chain CH3 regions resulted in further increasedformation of bispecific antibodies (C). The combination of clones withonly mutations in the CH3 region (D), less bispecific antibody wasdetected. Wild-type human IgG4 was used as a standard control for ELISA(column E).

Example 8: “Glu” Scanning of Human IgG1 CH3 Regions

Fourteen positions from the CH3 regions of the human IgG1 antibody werechosen to carry out a series of “Glu” scanning experiments. Criteria forchoosing these fourteen positions were essentially as described inExample 5. The positions chosen for the “Glu” scanning were numbered1-14 as shown in FIG. 10. All the mutants were generated by site-directmutagenesis kit from Stratagene.

The template used to generate the IgG1 mutants was Ab2.hFc1.EE, whichhas a wild-type IgG1 CH3 region, two mutations in the IgG1 hinge region,i.e., D221E and P228E.

All the mutants and the wild-type controls listed, as listed in FIG. 14Bwere expressed and purified individually. Equal amounts ofAb1.hFc1.RR.K409R IgG1 protein, which has a K409R mutation in the CH3region, two mutations in the hinge region, D221R and P228R, and variousAb2.hFc1.EE IgG1 CH3 mutants were mixed together and incubated with orwithout 0.5 mM GSH at 37° C. for 24 hour. Aliquots from each GSHreaction were diluted in ice-cold PBS-TB (PBS with 0.2% BSA, 0.05%Tween-20) and the amount of bispecific antibodies was measured bysandwich ELISA as described in Example 2. Ab1 and Ab2 with wild-typeIgG4 Fc region was expressed and treated in the same manner and used ascontrols in ELISA. The antibody Ab1 with a wild-type IgG4 Fc region andthe antibody 11A with a wild-type IgG4 Fc region were expressed andtreated in the same manner and used as controls in the ELISA assays.

As shown in FIG. 14B, very few positions in the CH3 domain of IgG1,i.e., Y349, L368, and F405, when substituted by Glu and combined with EE(K409R) mutant further increase bispecific antibody formation (columns2, 6, and 12).

Example 9: Comparison of Bispecific Antibody Formation

This Example illustrates the preparation of IgG1 hinge-containingheterodimers and compares bispecific antibody formation with otherisotypes.

All the mutants and the wild-type controls as shown in FIG. 15B wereexpressed and purified individually. Equal amount of antibody 1 andantibody 2 were mixed together and incubated with or without 0.5 mM GSHat 37° C. for 24 hour. Aliquots from each GSH reactions was diluted inice-cold PBS-TB (PBS with 0.2% BSA, 0.05% Tween-20) and the amount ofbispecific antibodies was measured by sandwich ELISA as describedbefore.

As shown in FIG. 15A, introducing mutations at two positions of the IgG1hinge region, D221 and P228, significantly affected levels of IgG1bispecific antibody formation. Removing the mutation in position D221dramatically decreased bispecific antibody formation, even when combinedwith a CH3 mutation (column 1). Within all three isotypes, IgG1 mutantsgenerate the greatest level of bispecific antibodies (compare columns 2,3 and 4). Standard controls were the same as in previous examples.

Example 10: Generation and Purification of Heterodimeric Antibodies

IgG1 heterodimers were prepared by incubation of Antibody 1 having 221R,228R, and 409R mutations with Antibody 2 having 221E, 228E, and 368Emutations in PBS with 1 mM GSH for 24 hrs at 37° C. Different antibodyvariable regions were used for preparation of the heterodimers, i.e.,Ab1, Ab2, Ab3, and Ab4. IgG2 heterodimers were prepared by incubation ofAntibody 1 having 223R, 225R, 228R, and 409R mutations with Antibody 2having 223E, 225E, 228E, and 368E mutations in PBS with 2 mM GSH for 24hrs at 37° C. The heterodimer was purified by ion exchangechromatography, as described below. IgG4 heterodimers were prepared byincubation of Antibody 1 with 228R mutation with Antibody 2 with 228Emutation in PBS with 1 mM GSH for 24 hrs at 37° C.

All the heterodimers were purified by ion exchange chromatography.Briefly, analytical ion exchange separation of the Fc-hetero andFc-homodimers was carried out on Agilent 1100 quaternary pump LC system(Agilent Inc, Santa Clara, Calif., USA) equipped with weak cationexchange DIONEX Propac WCX-10G (4×50 mm) column. Proteins were injectedin 5% buffer A (20 mM MES pH 5.4) and eluted in a gradient from 25% to75% buffer B (20 mM MES pH 5.4 and 500 mM NaCl) over a 20 minute periodwith 1 ml/min flow rate. Larger scale Fc-heterodimer purification wasperformed on an Akta Explorer (GE) equipped with weak cation exchangeDIONEX Propac WCX-10G (4×250 mm) column. Proteins were injected in 5%buffer A (20 mM MES pH 5.4) and eluted in a gradient from 15% to 75%buffer B (20 mM MES pH 5.4 and 500 mM NaCl) over a 60 minute period withlml/min flow rate. See FIGS. 16A-23C.

Example 11: Effect of CH3 Mutations on Heterodimer Formation

The example illustrates the effect of various CH3 and/or hinge mutationson heterodimeric protein formation.

a) CH3 Mutations at L368D, L368E, and K409R, and Wild-Type or MutantHinge

Plasmid vectors encoding the antibody mutants depicted in FIG. 24B wereprepared using the methods described above. Antibody Ab2 is ananti-antigen B antibody comprising a lambda light chain, and AntibodyAb1 is an anti-antigen A antibody comprising a kappa light chain. Inthis example, where mutations were made in the IgG1 hinge, the mutationswere at positions D221 and P228. Where mutations were made in the IgG2hinge, the mutations were at C223, E225 and P228. In this example, someof the mutants contained a CH3 mutation and a wild-type (wt) hinge.Other mutants contained mutations in both the hinge and the CH3 regions.In this example, the CH3 mutations were selected from K409R, L368D, andL368E.

The Group A and Group B mutants shown in FIG. 24B were expressed andpurified individually. Combination pairs 1-11 were tested for bispecificantibody formation. For each of the combinations 1-11, equal amounts ofthe specified Group A antibody and corresponding Group B antibody weremixed together and incubated with or without 0.5 mM GSH at 37° C. for 24hours. Aliquots from each GSH reactions was diluted in ice-cold PBS-TB(PBS with 0.2% BSA, 0.05% Tween-20) and the amount of bispecificantibodies was measured by sandwich ELISA. To detect bispecificantibodies, 0.25 μg/ml purified total proteins from each GSH reactionwere added onto an ELISA plate coated with 1 μg/ml antigen B. The amountof bispecific antibodies in each preparation was detected by HRPconjugated anti-kappa antibody.

As shown in FIG. 24A, introducing the K409R CH3 mutation in IgG1 wassufficient to promote some bispecific antibody formation (column 1). Incontrast, the L368E CH3 mutation alone did not result in a significantamount of bispecific antibody formation (column 2).

b) CH3 Mutations at L368E and/or K409R, and Wild-Type or Mutant Hinge

Plasmid vectors encoding the antibody mutants depicted in FIG. 25B wereprepared using the methods described above. In this example, wheremutations were made in the IgG1 hinge, the mutations were at positionsD221 and P228. Where mutations were made in the IgG2 hinge, themutations were at C223, E225, and P228. In this example, some of themutants contained a CH3 mutation and a wild-type (wt) hinge. Othermutants contained mutations in both the hinge and the CH3 regions. Inthis example, the CH3 mutations were selected from K409R and L368E.

The Group A and Group B mutants shown in FIG. 25B were expressed andpurified individually. Combination pairs 1-15 were tested for bispecificantibody formation using the methods described above in section (a).

hIgG1 and hIgG2 heterodimers were purified by ion exchangechromatography using the method described in Example 10. FIGS. 26A-26Dillustrate that the CH3 only mutation provides about 12% IgG1 or 13%IgG2 heterodimeric protein formation (mutations at K409R and L368E) incomparison to the wild type hIgG1 and that the combination of both thehinge (mutations at D221R, P228R, D221E, and P228E) and the CH3mutations (mutations at K409R and L368E) provides about 90% IgG1heterodimeric protein formation in comparison to the wild-type hIgG1heterodimeric protein.

Example 12: Differential Scanning Calorimetry Measuring the Stability ofthe Bispecific Antibody and its Parental Mutant Monospecific Antibodies

Differential Scanning calorimetry (DSC) measuring the stability of thebispecific antibodies was carried out for all antibody samples: 1)wild-type hIgG1 antibodies 5 and 6 (Ab5. wild-type hIgG1 andAb6.wild-type hIgG1); 2) parental hIgG1 antibody 5 with hinge mutations(D221E and P228E) and CH3 mutation (L368E) and parental hIgG1 antibody 6with hinge mutations (D228R and P228R) and CH3 mutation (K409R)(hIgG1.EE.L368E.Ab5.Ab5 or hIgG1.RR.K409R.Ab6.Ab6); and 3) bispecifichIgG1 antibody 5+6 with mutations at D221R, P228R, D221E, P228E, L368E,and K409R (hIgG1.EE.L368E.Ab5.Ab5/hIgG1.RR.K409R.Ab6.Ab6). Themeasurements were made at a concentration of 1.0 mg/mL at pH 7.2 in PBSbuffer on a MICROCAL™ VP capillary DSC system (GE Healthcare,Piscataway, N.J., USA). Samples were scanned at a rate of 90° C./hr from30 to 110° C. Data analysis was performed using OriginLab software(OriginLab Corporation, Northampton, Mass., USA).

The wild-type hIgG1 antibodies show melting temperature (Tm) of the CH3domain at about 86° C., while the parental hIgG1 mutant antibody 5 or 6has reduced Tm of 60° C. The Tm of the Ab6 mutant(hIgG1.RR.K409R.Ab6.Ab6/hIgG1.RR.K409R.Ab6.Ab6) appears similar to theFab domain with CH3 Tm at about 75° C. Upon formation of the bispecificantibody, the Tm of the CH3 domain is about 75° C. FIG. 27.

Example 13: Simultaneous Binding of Two Different Antigens by theBispecific Antibody

This example illustrates the ability of the heterodimeric proteinsdisclosed herein to simultaneously bind two different antigens.

Antigens A and B

A Biacore 3000 SPR biosensor instrument (GE Healthcare, Piscataway,N.J., USA) was used for this analysis. The (antigen A)-hFc antigen wascoupled to a Biacore CM5 sensor chip surface using an amine-couplingprocedure. The running buffer for the immobilization procedure wasHBS−T+(10 mM HEPES, 150 mM NaCl, 0.05% Tween-20, pH 7.4). The CM5 sensorsurface was activated by injecting a 1:1 (v/v) mixture of 400 mM EDC(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) and 100 mM NHS(N-Hydroxysuccinimide) for 7 minutes at 10 ul/min. Then, (antigen A)-hFcwas diluted to 50 μg/mL in 10 mM acetate buffer at pH 5.0 and injectedat 20 ul/min for 7 minutes. The surface was blocked by injecting 1 Methanolamine, pH 8.5 over the sensor surface at 10 ul/min.

After immobilization, first, 2 μg/mL bispecific antibody(hIgG1.EE.L368E.Ab1.Ab1/hIgG1.RR.K409R.Ab2.Ab2; mutation at hinge regionof D221R, P228R, D221E, and P228E and the CH3 region of K409R and L368E)was injected for 1 minute at 10 ul/min. Second, a “sandwiching analyte”was injected for 2 minutes at 10 ul/min. The “sandwiching analytes”tested were 972 nM antigen B, 1000 nM (antigen A)-ECD-his, and runningbuffer. The surfaces were regenerated with two 6-second injections of a2:1 (v/v) mixture of Pierce Elution Buffer:4M NaCl (Thermo FisherScientific, Rockford, Ill., USA).

FIG. 28A shows that the bispecific antibodyhIgG1EE.L368E.Ab1.Ab1/hlgG1RR.K409R.Ab2.Ab2 can simultaneously bindantigens A and B and that non-bispecific antibodies were not detected.

A similar experiment using antigen B coupling to a Biacore CM5 sensorchip surface was also conducted. All experimental conditions were thesame as the (antigen A)-hFc as described above, with the exception that2 ug/mL bispecific antibody was injected for 4 minutes, rather than 1minute, at 10 ul/min. The bispecific antibody was also able tosimultaneously bind antigens A and B.

Antigens C and D

A Biacore 3000 SPR biosensor instrument was also used for this analysis.The (antigen D)-hFc was coupled to a Biacore CM5 sensor chip surfaceusing an amine-coupling procedure. The running buffer for theimmobilization procedure was also HBS-T+. The CM5 sensor surface wasactivated by injecting a 1:1 (v/v) mixture of 400 mM EDC and 100 mM NHSfor 7 minutes at 10 ul/min. Then, (antigen D)-hFc was diluted to 30μg/mL in 10 mM sodium phosphate buffer at pH 6.5 and injected at 20ul/min for 7 minutes. The surface was blocked by injecting 1 Methanolamine at pH 8.5 over the sensor surface at 10 uL/min.

After immobilization, the running buffer was changed to HBS-T+ with 1mg/mL BSA (10 mM HEPES, 150 mM NaCl, 0.05% Tween 20, 1 mg/mL BSA, pH7.4). First, 1 μg/mL bispecific antibody(hIgG1EE.L368E.Ab4.Ab4/hlgG1RR.K409R.Ab3.Ab3; mutation at hinge regionof D221R, P228R, D221E, and P228E and the CH3 region of K409R and L368E)was injected for 2 minutes at 10 ul/min. Second, a “sandwiching analyte”was injected for 2 minutes at 10 ul/min. The “sandwiching analytes”tested were 20 nM antigen C, 200 nM (antigen-D)-ECD-his, and runningbuffer. The surfaces were regenerated with two 15-second injections of a2:1 (v/v) mixture of Pierce Elution Buffer:4M NaCl.

FIG. 28B shows that the bispecific antibodyhIgG1EE.L368E.Ab4.Ab4/hlgG1RR.K409R.Ab3.Ab3 can simultaneously bindantigens D and C and that non-bispecific antibodies were not detected.

A similar experiment using (antigen C)-hFc coupling to a Biacore CM5sensor chip surface was also conducted. All experimental conditions werethe same as the (antigen D)-hFc as described above, with the exceptionthat (antigen C)-hFc was diluted to 10 μg/mL in 10 mM acetate buffer atpH 5.0, rather than 30 ug/mL in 10 mM sodium phosphate buffer pH 6, andinjected at 20 ul/min for 7 minutes. The bispecific antibody was alsoable to simultaneously bind antigens C and D.

Example 14: Binding of the Bispecific Antibody to Fc-Gamma and FcRnReceptors

This example illustrates the ability of the heterodimeric proteinsdisclosed herein to bind Fc receptors.

Interaction analysis was conducted at 25° C. using a PROTEON™ XPR36surface plasmon resonance-based biosensor equipped with GLC sensor chipsand amine-coupling reagents (BioRad, Hercules, Calif.). The runningbuffer for the immobilizations and the analysis of theFc-gamma-receptors was PBS pH7.4+0.05% Tween-20. Buffer was flowed at 30uL/min. A panel of IgGs were amine-coupled onto separate “ligand”channels to levels of about 300-700 RU using a standardEDC/sulfo-NHS-mediated chemistry. The IgGs include bispecific hIgG1antibody 1+2 with mutations at D221R, P228R, D221E, P228E, L368E, andK409R (hIgG1.EE.L368E.Ab1.Ab1/hIgG1.RR.K409R.Ab2.Ab2); hIgG2 antibody1+2 with mutations at C223E, E225E, P228E, C223R, E225R, P228R, L368E,and K409R (hIgG2.EEE.L368E.Ab1.Ab1/hIgG2.RRR.K409R.Ab2.Ab2); and hIgG1and hIgG2 antibodies comprising a kappa light chain (Sigma-Aldrich, St.Louis, Mo., USA). Briefly, this involved activating for two minutes witha mixture of the stock solutions (supplied at 0.4M EDC and 0.1Msulfo-NHS) each diluted 1/600 in water, coupling the IgGs for threeminutes at 20 ug/mL in 10 mM sodium acetate at pH4.5, and finallydeactivating any excess reactive groups for three minutes with 1Methanolamine.HCl at pH8.5. The Fc-gamma-receptors were each prepared asa five-fold serial dilution with a variable top concentration, which wasoptimized per receptor (typically 200 nM for human Fc-gamma1 and 10 uMfor the other receptors). A five-membered serial dilution of eachreceptor including a buffer blank was injected in the “analyte”direction for three minutes in a “one-shot” mode, allowing up to 30minutes dissociation time. For receptors that did not dissociate fullywithin the allowed dissociation time, surfaces were regenerated at 100uL/min with two 18-sec injections of a 2:1 (v/v) mixture of PierceGentle Elution Buffer/4M NaCl (Thermo Scientific, Rockford, Ill., USA).Some receptors were injected in duplicate binding cycles to verify thatthe assay was reproducible.

The interactions of the immobilized IgGs with human-FcRn (neonatal Fcreceptor) were conducted in a different manner. The IgGs used are thesame bispecific hIgG1 and hIgG2 antibodies 1+2 as described above. Thecontrol antibody used is a human IgG2 ΔA. The IgGs were coupled ontoseparate reaction spots rather than channels (Abdiche et al, Anal.Biochem. 411(1):139-151 (2011)), the analysis running buffer wasPBS+0.05% Tween-20 pH6.0, and the human-FcRn was injected in a kinetictitration mode as both a five-fold and three-fold dilution series, eachwith a top concentration at 900 nM. Association and dissociation timeswere three and five minutes respectively and no regeneration wasrequired. Data processing and analysis were performed within thePROTEON™ Manager software v 2.1. Response data for each interaction weredouble referenced by subtracting the responses from the interspots(unmodified chip) and the responses from the buffer blanks, and then fitglobally to a simple Langmuir kinetic model. The equilibriumdissociation constant (K_(D)) was deduced from the ratio of the kineticrate constants (K_(D)=k_(d/)k_(a)). For interactions that rapidlyreached equilibrium binding responses within the association phase, theK_(D) was deduced via an equilibrium binding model.

Table 4 shows that binding of IgG1 and IgG2 bispecific antibodies toFc-gamma (Fcγ) receptors is similar to the control hIgG1 and hIgG2antibodies. Table 5 shows that FcRn binding of IgG1 and IgG2 bispecificantibodies is also similar to the control hIgG2ΔA antibody.

TABLE 4 Fcγ receptors (in solution) IgG coupled hFcγ1 hFcγ2A hFcγ2BhFcγ3A hFcγ3B mFcγ1 mFcγ2B mFcγ3 hIgG1 kappa (control) 0.2 nM weak  3 uM272 nM weak 120 nM weak weak hIgG1.EE.L368E.Ab1.Ab1/ 0.4 nM weak  6 uM671 nM weak 260 nM weak weak hIgG1.RR.K409R.Ab2.Ab2 hIgG2 kappa(control) none very weak >10 uM 1930 nM  very weak none very weak veryweak hIgG2.EEE.L368E.Ab1.Ab1/ none very weak >10 uM none none none nonenone hIgG2.RRR.K409R.Ab2.Ab2

TABLE 5 IgG coupled Kd (uM)hIgG1.EE.L368E.Ab1.Ab1/hIgG1.RR.K409R.Ab2.Ab2 0.826hIgG2.EEE.L368E.Ab1.Ab1/hIgG2.RRR.K409R.Ab2.Ab2 0.60 hIgG2ΔA (control)0.983

Example 15: In Vitro Growth Inhibition Assay and Off-Rate Measurement ofthe Bispecific Antibody

This example illustrates the ability of a heterodimeric protein toinhibit cell growth in vitro.

In vitro activity of the Ab3+Ab4 IgG1 bispecific antibody on cell growthin comparison to its parental bivalent monospecific antibodies as wellas their monovalent counterparts was determined.

Growth Inhibition Assay

Cal27 tongue carcinoma cells or FaDu head and neck carcinoma cells wereseeded at 3000 cells/well in RPMI 1640 medium+2% FBS (fetal bovineserum) and grown in the 96-well plate overnight. A serial dilution ofantibodies in RPMI 1640 medium+2% FBS was then added to each well andcells were allowed to grow for 5 days at 37° C. At the end of the assay,the amount of cells was measured by the Cell Titer Glo kit (Promega,Madison, Wis., USA) as per manufacturer's protocol. The amount of cellsfor each antibody concentration was normalized to that of control humanIgG1 treatment and used to generate the dose-response curve. All sampleswere performed in triplicate.

Cell-Based Antibody Dissociation Rate Constant Measurement

Cal27 tongue carcinoma cells were grown on poly-D-lysine coated 96-wellplate in DMEM+10% FBS until near confluent. Wells were washed with PBSand followed by 2% paraformaldehyde fixation for 15 minutes at roomtemperature. All subsequent incubation was done at room temperature. Forimmunofluorescent staining, wells were blocked with DMEM/B (DMEM+5% BSA)for 1 hour. Dylight800-labeled (labeling kit from Thermo Scientific,Rockford, Ill., USA) target-specific antibodies diluted in DMEM/B wereadded to wells and incubated for 1 hour. Wells were then washed threetimes with 250 ul DMEM/B. To measure antibody-antigen dissociation, 150ul of 50 ug/ml unlabeled target-specific antibodies was added to eachwell (except those for timepoint “0”) and incubated at room temperaturefor various time for up to 21 hours. At the end of incubation, antibodysolution was discarded and replaced with 100 ul of 10 uM DRAQ5™(Biostatus Limited, United Kingdom) and further incubated for 8 minutes.Subsequently, DRAQ5™ solution was discarded, and wells were air-driedwhile protected from light. For timepoint “0”, wells were directlystained with DRAQ5™ without incubation with unlabeled antibodies. Allsamples were done in triplicate.

Plate was then read by Li-Cor ODYSSEY® infrared imaging system (LI-CORBiotechnology, Lincoln, Nebr.) to measure the fluorescent intensity at800 nm, which corresponded to the amount of Dylight800-labeled antibodyremained bound on cell surface, and 700 nm (DRAQ5), which stained DNAand hence correlated with the number of cells in each well. For eachwell, the fluorescent intensity at 800 nm was normalized by the value at700 nm to account for the well-to-well variation of total cell number.Subsequently, the normalized fluorescent intensity for each well wasnormalized again by the corresponding value at time point “0” and thenplotted against dissociation time to generate an exponential decaycurve. The curve was then fitted to a single exponential decay equationusing GraphPad Prism to generate the apparent dissociation rateconstant.

Ab3/Ab4 Bispecific Antibody Effectively Inhibits Growth of Cal27 andFaDu Cells

To investigate the in vitro activity of Ab3/Ab4 bispecific antibody oncell growth, the bispecific antibody was compared to their parentalbivalent, monospecific antibodies as well as their monovalentcounterparts. As shown in FIG. 29, monovalent Ab4/nc.biFc(hIgG1.RR.K409R.Ab4.Ab4/hIgG1.EE.L368E.Ab6.Ab6; mutations at D221R,P228R, D221E, P228E, L368E, and K409R) as well as bivalent Ab4.hIgG1(Ab4.wild-type hIgG1) had no significant effect on cell growth acrossall concentrations tested while monovalent Ab3/nc.Fc(hIgG1.RR.K409R.Ab3.Ab3/hIgG1.EE.L368E.Ab6.Ab6; mutations at D221R,P228R, D221E, P228E, L368E, and K409R) inhibited (>10%) growth of Cal27and FaDu cells at concentration >1 ug/ml. Yet, when the negative control(nc) arm of the monovalent Ab3/nc.biFc antibody was replaced by Ab4 togenerate the bispecific antibody(hIgG1.EE.L368E.Ab3.Ab3/hIgG1.RR.K409R.Ab4.Ab4), it significantlyaugmented the growth inhibitory activity of the Ab3 arm to a level thatwas comparable to the bivalent monospecific Ab3 antibodies (Ab3.hIgG1and Ab3.biFc). This effect is hypothesized due to gain in avidity as aresult of binding of the Ab4 arm to its cell surface target and thusincreases the local concentration of Ab3 on cell surface and hence theoccupancy of Ab3 target. FIG. 29.

Ab3/Ab4 Bispecific Antibody has a Slower Apparent Dissociation RateConstant than its Monovalent Counterparts

To obtain evidence of avidity gain in the bispecific antibody, theapparent dissociation rate constants of the bispecific antibody and itsmonovalent counterparts were measured on Cal27 cells. As shown in FIG.30, the apparent dissociation rate constant of Ab3/Ab4.biFc(hIgG1.EE.L368E.Ab3.Ab3/hIgG1.RR.K409R.Ab4.Ab4) was about 2-fold slowerthan that of the monovalent antibodies, Ab3/nc.biFc(hIgG1.RR.K409R.Ab3.Ab3/hIgG1.EE.L368E.Ab6.Ab6) and Ab4/nc.biFc(hIgG1.RR.K409R.Ab4.Ab4/hIgG1.EE.L368E.Ab6.Ab6). Taken together, thedata suggest that the bispecific antibody gained avidity through bindingof both Ab3 and Ab4 arms to cell surface.

Example 16: In Vivo Efficacy Studies of the Bispecific Antibody onTarget-Expressing Cell Xenograft Models

This example illustrates the in vivo efficacy of heterodimeric proteinsprepared using the methods described herein.

In vivo efficacy studies of bispecific antibodies are performed ontarget-expressing cell xenograft models compared to wild-type bivalentmonospecific antibodies. More specifically, subcutaneous tumor growthcurves (representing tumor types including, but not limited to,pancreatic, head and neck, colon, gastric, breast, prostate or lungcancer) in immunodeficient nu/nu or SCID (Severely CombinedImmunodeficient) mice are established prior to efficacy studies toobtain optimal cell numbers for tumor implantation. A typical efficacyis carried out in the following steps: 1) Tumor cells are implantedsubcutaneously into 5-8 weeks old immunodeficient mice until the tumorsizes reach 50-100 mm³, 2) Dosing is done through bolus tail veininjection. Depending on the tumor response to treatment, animals areinjected with 1-100 mg/kg of bispecific (e.g.,hIgG1.EE.L368E.Ab4.Ab4/hlgG1.RR.K409R.Ab3.Ab3; mutations at D221R,P228R, D221E, P228E, L368E, and K409R) or wild-type antibodies (hIgG1Ab3 or hIgG1 Ab4) up to three times a week. 3) Dosing continues untilthe tumor sizes in the control group reach 2000 mm³. All experimentalanimals are monitored for body weight changes daily. Tumor volume ismeasured twice a week by a Caliper device and calculated with thefollowing formula: Tumor volume=(length×width²)/2. Efficacy is expressedas the percentage tumor growth inhibition (% TGI); calculated using theequation 100−(T/C×100), where T is the MTV (median tumor volume) of thetreatment group and C is the MTV of the control group. The bispecificantibodies are as efficacious as the wild-type bivalent monospecificantibodies in tumor growth inhibition. Further, with reduced affinity tonormal tissues, the MTD (maximum tolerated doses) for bispecificantibodies is higher, thereby resulting in greater Therapeutic Indicesdefined as maximum tolerated dose/minimum curative dose.

Example 17: In Vivo Study of the Bispecific Antibody on T-Cell MediatedKilling of CD20 Positive B Cells

This example illustrates the in vivo efficacy of the bispecific antibodyas described herein on T-cell mediated killing of CD20 positive B cells.

The full-length bispecific antibodies (IgG2ΔA) that are specific tomouse CD20 and CD3 (e.g.,hIgG2.EEE.L368E.CD3.CD3/hIgG2.RRR.K409R.CD20.CD20 (mutations at C223E,E225E, P228E, C223R, E225R, P228R, L368E, and K409R)) were generatedusing the methods described herein. A dose response experiment was donein wild-type C57/Bl6 mice, and CD19 positive lymphocytes were measuredin peripheral blood 5 days after a single intravenous dose of thebispecific CD3/CD20 antibody. Doses of 200 μg/kg or greater effectivelydepleted the population of CD19 positive lymophocytes. See Table 6.

TABLE 6 CD19 (+) lymphocytes (%) Dose Pre-bleed Day 5 PBS 40.9 41.8 PBS31.5 46.2 PBS 51.4 37 PBS 36.9 30.1 PBS 43.9 35.1 8 μg/kg 43.9 39.1 8μg/kg 41.1 38.8 8 μg/kg 37.5 25.8 40 μg/kg 45.4 32.7 40 μg/kg 37.3 28.640 μg/kg 51 42.7 200 μg/kg 43.1 3.49 200 μg/kg 48.6 1.56 200 μg/kg 41.50.74 1 mg/kg 47.5 0 1 mg/kg 50.5 0 1 mg/kg 37.6 0 5 mg/kg 44.5 0 5 mg/kg37.2 0 5 mg/kg 43 0

Example 18 In Vitro Study of the Bispecific Antibodies on T-CellMediated Killing of EpCAM Positive Tumor Cells

This example illustrates the ability of a heterodimeric protein to killtumor cells mediated by cytotoxic T cells in vitro.

The full-length human bispecific IgG2ΔA antibody specific to EpCAM andCD3 (e.g., hIgG2.EEE.L368E.EpCAM.EpCAM/hIgG2.RRR.K409R.CD3.CD3(mutations at C223E, E225E, P228E, C223R, E225R, P228R, L368E, andK409R)) were generated using the methods described herein. The efficacyof the bispecific EpCAM/CD3 antibody was determined by using the cellkilling assay set at different effector and target cell ratio (e.g., E/T5 and E/T 10) and a 4-day time course (e.g., measured at 24, 48, 72, 96hours). The EpCAM positive tumor cells (SW480) were used as the targetcells and the PBMC (peripheral blood mononuclear cells) were isolatedfrom healthy donor blood as effector cells. The cytotoxic potential ofthe bispecific EpCAM/CD3 antibody was assessed by CYTOTOX96®Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis., USA). FIGS.31A and 31B show that the bispecific EpCAM/CD3 antibody generated inthis example induced the killing of EpCAM positive tumor cells (SW480).More specifically, after co-culturing the SW480 cells with PBMC for atleast 72 hours, significant lysis of the SW480 cells was observed afterthe addition of the bispecific EpCAM/CD3 antibody (labeled as“hG2-EpCAM-CD3” in the figures) at 10 nM. The SW480 cells werequantitatively killed at a concentration greater than 200 nM. Similarcell-lysis results using the bispecific EpCAM/CD3 IgG1 antibody (e.g.,hIgG1.EE.L368E.EpCAM.EpCAM/hlgG1.RR.K409R.CD3.CD3; mutations at D221R,P228R, D221E, P228E, L368E, and K409R) were also observed.

Although the disclosed teachings have been described with reference tovarious applications, methods, and compositions, it will be appreciatedthat various changes and modifications can be made without departingfrom the teachings herein and the claimed invention below. The foregoingexamples are provided to better illustrate the disclosed teachings andare not intended to limit the scope of the teachings presented herein.While the present teachings have been described in terms of theseexemplary embodiments, the skilled artisan will readily understand thatnumerous variations and modifications of these exemplary embodiments arepossible without undue experimentation. All such variations andmodifications are within the scope of the current teachings.

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated byreference in their entirety. In the event that one or more of theincorporated literature and similar materials differs from orcontradicts this application, including but not limited to definedterms, term usage, described techniques, or the like, this applicationcontrols.

The foregoing description and Examples detail certain specificembodiments of the invention and describes the best mode contemplated bythe inventors. It will be appreciated, however, that no matter howdetailed the foregoing may appear in text, the invention may bepracticed in many ways and the invention should be construed inaccordance with the appended claims and any equivalents thereof.

What is claimed is:
 1. A cell line expressing a heterodimeric protein,wherein the heterodimeric protein comprises: a hinge region comprising afirst immunoglobulin-like hinge polypeptide and a secondimmunoglobulin-like hinge polypeptide which interact together to form adimeric hinge interface, wherein electrostatic interactions between oneor more charged amino acids within the hinge interface favor interactionbetween the first and second hinge polypeptides over interaction betweentwo first hinge polypeptides or two second hinge polypeptides, therebypromoting heterodimer formation over homodimer formation, wherein (a)the hinge region is a human IgG1 hinge region, wherein each of the firsthinge polypeptide and the second hinge polypeptide comprises amino acidmodifications relative to a wild-type IgG1 hinge region at a position ofAsp221 and Pro228, according to EU numbering scheme, wherein thewild-type amino acid in the first hinge polypeptide is replaced with anamino acid having an opposite charge to the corresponding amino acid inthe second hinge polypeptide, and the heterodimeric protein furthercomprising an immunoglobulin-like CH3 region comprising a first CH3polypeptide fused to the first hinge polypeptide and a second CH3polypeptide fused to the second hinge polypeptide, wherein the first CH3polypeptide and the second CH3 polypeptide comprise at least one aminoacid modification relative to a wild-type IgG1 CH3 region sequence at aposition of Tyr349, Leu368, Phe405, or Lys409, according to EU numberingscheme; or (b) the hinge region is a human IgG2 hinge region, whereineach of the first hinge polypeptide and the second hinge polypeptidecomprises at least two amino acid modifications relative to a wild-typeIgG2 hinge region at a position of Cys223, Glu225 or Pro228, accordingto EU numbering scheme, wherein the wild-type amino acid in the firsthinge polypeptide is replaced with an amino acid having an oppositecharge to the corresponding amino acid in the second hinge polypeptide,and the heterodimeric protein further comprising an immunoglobulin-likeCH3 region comprising a first CH3 polypeptide fused to the first hingepolypeptide and a second CH3 polypeptide fused to the second hingepolypeptide, wherein the first CH3 polypeptide and the second CH3polypeptide comprise at least one amino acid modification relative to awild-type IgG2 CH3 region sequence at a position of Leu368 or Lys409,according to EU numbering scheme.
 2. The cell line of claim 1, whereinthe hinge region of the heterodimeric protein is a human IgG2 hingeregion, the first hinge polypeptide comprises the amino acidmodification of Cys223Arg, Glu225Arg and Pro228Arg, the second hingepolypeptide comprises the amino acid modification of Cys223Glu andPro228Glu, and wherein (i) the first CH3 polypeptide comprises the aminoacid modification of Lys409Arg and the second CH3 polypeptide comprisesthe amino acid modification of Leu368Glu or Leu368Asp, (ii) the firstCH3 polypeptide comprises the amino acid modification of Leu368Glu orLeu368Asp, and the second CH3 polypeptide comprises the amino acidmodification of Lys409Arg, or (iii) each of the CH3 polypeptidecomprises the amino acid modification of Lys409Arg.
 3. The cell line ofclaim 2, wherein the first CH3 polypeptide comprises the amino acidmodification of Lys409Arg and the second CH3 polypeptide comprises theamino acid modification of Leu368Glu.
 4. The cell line of claim 2,wherein the heterodimeric protein is a full length human IgG2 antibody.5. The cell line of claim 2, wherein the heterodimeric protein is a fulllength human IgG2 delta A antibody comprising two heavy chains, andwherein each heavy chain comprises an amino acid modification ofAla330Ser and Pro331Ser, according to EU numbering scheme.
 6. The cellline of claim 1, wherein the hinge region is a human IgG1 hinge region,and wherein the first hinge polypeptide comprises the amino acidmodification of Asp221Arg and Pro228Arg, the second hinge polypeptidecomprises amino acid modification of Asp221Glu and Pro228Glu, andwherein (i) the first CH3 polypeptide comprises the amino acidmodification of Lys409Arg and the second CH3 polypeptide comprises theamino acid modification of Leu368Glu or Leu368Asp, or (ii) each of theCH3 polypeptide comprises the amino acid modification of Lys409Arg. 7.The cell line of claim 6, wherein the first CH3 polypeptide comprisesthe amino acid modification of Lys409Arg and the second CH3 polypeptidecomprises the amino acid modification of Leu368Glu.
 8. The cell line ofclaim 6, wherein the heterodimeric protein is a full length human IgG1antibody.
 9. A method of producing the heterodimeric protein of claim 1,comprising the steps of: a) culturing a host cell comprising a nucleicacid encoding the first hinge and CH3 polypeptides and a nucleic acidencoding the second hinge and CH3 polypeptides, wherein the culturedhost cell expresses the first and second hinge and CH3 polypeptides; andb) optionally, recovering the heterodimeric protein from the host cellculture.
 10. A method of producing the heterodimeric protein of claim 1,comprising the steps of: a) expressing the first hinge and CH3polypeptides in a first host cell; b) expressing the second hinge andCH3 polypeptides in a second host cell; c) optionally, isolating thefirst hinge and CH3 polypeptides and the second hinge and CH3polypeptides; and d) incubating the first and second hinge and CH3polypeptides under a condition suitable for dimerization to produce theheterodimeric protein.
 11. The method of claim 10, wherein the conditionof step d) is to incubate the two hinge and CH3 polypeptides of step c)with a reducing agent.
 12. The method of claim 11, wherein the reducingagent is glutathione.